Compositions and methods for inhibiting gene expression of alpha-1 AntiTrypsin

ABSTRACT

The invention relates to a RNA interference triggers for inhibiting the expression of an AAT gene through the mechanism of RNA interference. The invention also relates to a pharmaceutical composition comprising the AAT RNAi trigger together with an excipient capable of improving delivery of the RNAi trigger to a liver cell in vivo. Delivery of the AAT RNAi trigger to liver cells in vivo provides for inhibition of AAT gene expression and treatment of alpha 1-antitrypsin deficiency and associated diseases.

BACKGROUND OF THE INVENTION

Alpha-1 antitrypsin deficiency is an inherited autosomal codominantgenetic disorder that causes defective production of alpha 1-antitrypsin(A1AT) leading to lung and liver diseases and occurs with frequencyabout 1 case in 1,500 to 3,500 individuals. Alpha-1 antitrypsindeficiency most often affects persons with European ancestry worldwide.

Alpha-1 Antitrypsin (α1-antitrypsin, alpha-1 proteinase inhibitor, A1AT,or AAT) is a protease inhibitor belonging to the serpin superfamily.Normal AAT protein is primarily synthesized in the liver by hepatocytesand secreted into blood. Its physiologic function is to inhibitneutrophil proteases in order to protect host tissues from non-specificinjury during periods of inflammation. The most clinically significantform of A1AT deficiency (AATD) is caused by the Z mutation. The Z mutantallele (PiZ), through a single point mutation, renders the mutant PiZprotein prone to abnormal folding in the endoplasmic reticulum ofhepatocytes causing intracellular retention. The absence of circulatinganti-protease activity leaves the lung vulnerable to injury byneutrophil elastase, resulting in the development of emphysema. Weeklyuse of AAT augmentation therapy for AATD, using purified human AAT,results in normal plasma levels of AAT and prevents lung damage inaffected individuals.

While administration of purified AAT ameliorates lung damage caused bythe absence of endogenously secreted AAT, AATD patients remainvulnerable to endoplasmic reticulum liver storage disease caused by thedeposition of excessive abnormally folded AAT protein. Twelve to fifteenpercent of patients with AATD also develop liver disease, which can besevere or fatal, even in infancy. The intracellular accumulation inhepatocytes of AAT protein in AATD patients induces liver cell damageand death, and chronic liver injury. Clinical presentations includechronic hepatitis, cirrhosis, hepatocellular carcinoma, transaminitis,cholestasis, fibrosis, and even fulminant hepatic failure.

There is currently no specific treatment to prevent the onset or slowthe progression of liver disease due to AATD. Because liver damageresulting from AATD occurs through a gain-of-function mechanism,inhibition or AAT gene expression would be useful in preventingaccumulation of the AAT protein in the liver, thereby providing atherapeutic treatment for AATD. Double-stranded RNA molecules (dsRNA)and other RNAi triggers have been shown to block gene expression in ahighly conserved regulatory mechanism known as RNA interference (RNAi).The invention provides AAT RNA interference (RNAi) triggers andcompositions thereof for inhibiting the expression of the AAT gene invivo. The invention also provides methods of using the AAT RNAi triggersfor treating AATD and conditions and diseases caused by AATD, such aschronic hepatitis, cirrhosis, hepatocellular carcinoma, and fulminanthepatic failure.

SUMMARY OF THE INVENTION

The invention provides alpha-1 antitrypsin (AAT) gene specific RNAinterference (RNAi) trigger molecules able to selectively andefficiently decrease expression of AAT. The use of AAT RNAi triggerprovides a method for the therapeutic treatment of diseases associatedwith alpha-1 antitrypsin deficiency. Such methods compriseadministration of RNAi trigger targeting AAT to a human being or animal.

In one embodiment, the invention provides RNAi trigger molecules forinhibiting expression of the human AAT gene. The RNAi trigger comprisesat least two sequences that are partially, substantially, or fullycomplementary to each other. In one embodiment, the two RNAi triggersequences comprise a sense strand comprising a first sequence and anantisense strand comprising a second sequence. In another embodiment,the two RNAi trigger sequences comprise two sense strands which togethercomprise a first sequence and an antisense strand comprising a secondsequence, wherein the sense strands and the antisense strand togetherform a meroduplex (Tables 2 and 4). The AAT RNAi trigger sense strandscomprise sequences which have an identity of at least 90% to at least aportion of an AAT mRNA. Exemplary AAT RNAi trigger sense strands,antisense strands, sequence pairs and meroduplexes are shown in Tables1-5.

In one embodiment, the antisense strand comprises a nucleotide sequencewhich is complementary to a part of an mRNA encoded by said AAT gene,and the region of complementarity is most preferably less than 30nucleotides in length. Furthermore, it is preferred that the length ofthe herein described inventive RNAi triggers (duplex length) is in therange of about 16 to 30 nucleotides, in particular in the range of about18 to 28 nucleotides. Particularly useful in context of this inventionare duplex lengths of about 17, 18, 19, 20, 21, 22, 23 or 24nucleotides. The sense and antisense strands can be the same length orthey can be different lengths. For example, both the sense and antisensestrands can be 19, 20, 21, 22, 23, or 24 nucleotides in length. As anexample, the sense strand can be 21 nucleotides in length while theantisense strand is 23 nucleotides in length. Most preferred are duplexstretches of 19, 21, 22, or 23 nucleotides. The RNAi trigger, upondelivery to a cell expressing the AAT gene, inhibits the expression ofsaid AAT gene in vitro or in vivo.

The RNAi trigger molecules or pharmaceutical compositions describedherein can be administered in a number of ways depending upon whetherlocal or systemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer: intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The RNAi trigger molecules described herein can be delivered to targetcells or tissues using any known oligonucleotide delivery technologyknown in the art. Nucleic acid delivery methods include, but not limitedto, encapsulation in liposomes, by iontophoresis, or by incorporationinto other vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres, proteinaceous vectors orDPCs (WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO2012/083185, each of which is incorporated herein by reference). In oneembodiment, the AAT RNAi trigger is provided with an in vivo deliverycompound. A preferred in vivo delivery compound comprises an MLPdelivery polymer.

In another preferred embodiment, the invention features a compositionfor delivering an AAT RNAi trigger to a liver cell in vivo comprising:an AAT RNAi trigger described herein conjugated to a hydrophobic groupcontaining at least 20 carbon atoms (RNA trigger-conjugate), such as acholesterol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Table 1. Core sequences of RNAi triggers targeting AAT mRNA.

FIG. 2 Table 2. RNAi trigger sequences containing 5′ and 3′ extensions.Letters in capitals represent ribonucleotides, lower case letters “c”,“g”, “a” and “u” represent 2′ O-methyl-modified nucleotides, upper caseletters A, C, G, U followed by “f” indicates a 2′-fluoro nucleotide, “s”represents phosphorothioate, “dT” represents deoxythymidine, (invdT)represents an inverted deoxythimidine (3′-3′-linked).

FIG. 3. Table 3. Canonical AAT siRNA RNAi triggers. Letters in capitalsrepresent RNA nucleotides, lower case letters “c”, “g”, “a” and “u”represent 2′ O-methyl-modified nucleotides, upper case letters A, C, G,U followed by “f” indicates a 2′-fluoro nucleotide, “s” representsphosphorothioate, “dT” represents deoxythymidine, and (invdT) representsan inverted deoxythimidine (3′-3′-linked). T_(m) is the meltingtemperature of the RNAi trigger.

FIG. 4. Table 4. AAT meroduplex RNAi triggers. Letters in capitalsrepresent RNA nucleotides, lower case letters “c”, “g”, “a” and “u”represent 2′ O-methyl-modified nucleotides, upper case letters A, C, G,U followed by “f” indicates a 2′-fluoro nucleotide, “s” representsphosphorothioate, “dT” represents deoxythymidine, and (invdT) representsan inverted deoxythimidine (3′-3′-linked). T_(m) is the meltingtemperature of the RNAi trigger.

FIG. 5. Table 5. AAT UNA RNAi triggers. Letters in capitals representRNA nucleotides, lower case letters “c”, “g”, “a” and “u” represent 2′O-methyl-modified nucleotides, upper case letters A, C, G, U followed by“f” indicates a 2′-fluoro nucleotide, upper case letters A, C, G, Ufollowed by “_(UNA)” indicates an 2′,3′-seco (unlocked) RNA nucleotidemimic, “s” represents phosphorothioate, “dT” represents deoxythymidine,and (invdT) represents an inverted deoxythimidine (3′-3′-linked). T_(m)is the melting temperature of the RNAi trigger.

FIG. 6A-E. Table listing MLP polymers suitable for use in delivery ofAAT RNAi triggers described herein in vivo.

FIG. 7. Graph depicting relative AAT expression in Hep3B cells in vitrousing varying concentrations of the indicated RNAi triggers.

FIG. 8. Graph depicting relative serum AAT levels in PiZ mice treatedsaline or SEQ ID 52/63 AAT RNAi trigger (AD00370) and MLP deliverypolymer (MLP).

FIG. 9. PAS-D staining to visualize Z-AAT accumulation in liver. Liversections from (A) PiZ mouse sacrificed at day 1 of study; (B) PiZ mousereceiving four biweekly IV doses of saline vehicle; (C) PiZ mousereceiving four biweekly IV doses of 8 mg/kg Luc-RNAi trigger control+8mg/kg MLP delivery polymer; (D) PiZ mouse receiving four biweeklyintravenous (IV) doses of 8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLPdelivery polymer.

FIG. 10. Western blot analysis of the soluble and insoluble fractionsfrom livers of PiZ mice. Five-week old mice received biweekly IV dosesof saline, Luc-UNA (8 mg/kg SEQ ID 59/78 with 8 mg/kg of MLP deliverypolymer), or AAT-UNA (8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLP deliverypolymer) for 8 weeks.

FIG. 11. Western blot analysis of the soluble and insoluble fractionsfrom livers of PiZ mice. Five-week old mice received four biweekly IVdoses of saline, Luc-UNA (8 mg/kg SEQ ID 59/78 with 8 mg/kg of MLPdelivery polymer), or AAT-UNA (8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLPdelivery polymer) for 8 weeks.

FIG. 12. Bar graph showing globule size in six month old female PiZ micethat received a single IV dose of saline, Luc-UNA RNAi trigger (8 mg/kgSEQ ID 59/78 with 8 mg/kg of MLP delivery polymer) or AAT-UNA RNAItrigger (8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLP delivery polymer). Thelivers were sectioned, processed in formalin for histologicalobservation, and stained with PAS-D for digital quantitation of theglobule size and the area of the liver covered by globules.

FIG. 13. Graph showing AAT knockdown following repeat administration inprimates with AAT-RNAi trigger and MLP delivery polymer. Two monkeyseach were given 2.0 mg/kg MLP delivery polymer (MLP deliverypeptide)+4.0 AAT-RNAi trigger SEQ ID 52/63 (AAT-UNA) or 3 mg/kg MLPdelivery polymer (MLP delivery peptide)+6 mg/kg RNAi trigger SEQ ID52/63 (AAT-UNA). The first dose was at day 1. Doses were all six weeksapart.

DETAILED DESCRIPTION OF THE INVENTION

Appended Tables 1-5 relate to preferred molecules and sequences to beused in forming AAT RNAi trigger molecules in accordance with theinvention. An AAT RNAi trigger molecule described herein comprises oneor two sense strands and an antisense strand each containing a coresequence of about 18 nucleobases. The antisense strand core sequence iscomplementary to a nucleotide sequence (target sequence) present in theAAT mRNA. The sense strand core sequence can be the same length as theantisense core sequence or it can be a different length. The sense andantisense core sequences of the RNAi triggers anneal to form acomplementary duplex region or double helical structure. Within thecomplementary duplex region, the sense strand core sequence is at least90% complementary or 100% complementary to the antisense core sequence.For meroduplex RNAi triggers, the sense strand core sequence isinternally nicked, and two sense strand sequences are provided thattogether hybridize with the antisense strand core sequence. In addition,the sense strands and antisense strands may independently containextensions of 1-6 nucleobases at the 5′ ends of their core sequences, 3′ends of their core sequences, or both the 5′ and 3′ ends of their coresequences. The antisense strand extensions, if present, may or may notbe complementary to the corresponding nucleotides for the AAT mRNA or toany corresponding nucleotides of the sense strand. Similarly, sensestrand extensions, if present, may or may not be identical to thecorresponding nucleotides for the AAT mRNA or to any correspondingnucleotides of the antisense strand. When delivered to a cell, the AATRNAi triggers described herein “knockdown” or inhibit expression of thenormal or Z mutant allele AAT gene.

The described AAT RNAi triggers and methods can be used to treat asubject having a disease or disorder that would benefit from reductionor inhibition in AAT expression. The subject is administered atherapeutically effective amount of any one or more of the described AATRNAi triggers. Treatment of a subject that would benefit from areduction and/or inhibition of AAT gene expression includes therapeuticand/or prophylactic treatment. The subject can be a human, patient, orhuman patient. The described AAT RNAi trigger molecules can be used toprovide a method for the therapeutic treatment of diseases associatedwith mutant AAT expression. Such methods comprise administration of RNAitrigger targeting AAT to a human being or animal.

AAT RNAi trigger sense and antisense strand core sequences are shown inTable 1. Table 2 provides for illustrative examples of RNAi triggersense and antisense strands described herein having 5′ or 3′ extensions.RNAi trigger sense and antisense strands having modified nucleotides areprovided herein and are in particular disclosed in appended Tables 3-5,providing illustrative examples of modified RNAi trigger sense andantisense strands of the present invention. The relation of the modifiedRNAi trigger strands shown in Tables 2-5 to the unmodified coresequences shown in Table 1 are indicated by the core SEQ ID numbers. Themodifications of these constituents of the inventive RNAi triggers areprovided herein as examples of modifications and/or modificationpatterns.

RNAi triggers (also called dsRNAi triggers) inhibit gene expressionthrough the biological process of RNA interference (RNAi). RNAi triggerscomprise double stranded RNA or RNA-like structures typically containing15-50 base pairs and preferably 18-26 base pairs and having a nucleobasesequence at least 90% complementary to a coding sequence in an expressedtarget gene within the cell. RNAi triggers include, but are not limitedto: short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), microRNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates (U.S.Pat. No. 8,084,599 8,349,809 and 8,513,207).

An RNAi trigger described here is formed by annealing an antisensestrand with a sense strand, for canonical siRNA RNAi triggers and UNARNAi triggers, or two sense strands, for meroduplex RNAi triggers. In apreferred embodiment, the AAT RNAi trigger antisense strands comprisenucleic acid sequences depicted in SEQ ID Nos: 1, 2, 3, 4, and 5. Thecorresponding AAT RNAi trigger sense strands comprise nucleic acidsequences depicted in SEQ ID Nos: 8, 9, 10, 11, and 12. Accordingly, theinventive AAT RNAi trigger molecules may, inter alia, comprise thesequence pairs selected from the group consisting of SEQ ID pairs: 1/8,2/9, 3/10, 4/11, and 5/12. Complementary pairs or meroplexes (RNAitriggers) are provided in Tables 1-5 as indicated by SEQ ID pair or SEQID mero.

As detailed below, the herein described RNAi trigger molecule sensestrands and antisense strands each comprises a core sequence andoptionally a 5′ extension, a 3′ extension, or a 5′ extension and a 3′extension. As used herein, an extension comprises 1-5 nucleotides at the5′ or 3′ end of the sense strand core sequence or antisense strand coresequence. The extension nucleotides on a sense strand may or may not becomplementary to (base pair with) nucleotides, either core sequencenucleotides of extension nucleotides, in the corresponding antisensestrand. Conversely, the extension nucleotides on an antisense strand mayor may not be complementary to (base pair with) nucleotides, either coresequence nucleotides of extension nucleotides, in the correspondingsense strand.

In one embodiment an AAT RNAi trigger molecule described hereincomprises an antisense strand with a 3′ extension of 1-5 nucleotides inlength, preferably 1-2 nucleotides in length. In one embodiment, one ormore of the antisense strand extension nucleotides comprise uracil orthymidine nucleotides or nucleotides which are complementary to thecorresponding AAT mRNA sequence. In a another embodiment, the antisensestrand extension consists of dTdT or dTsdT, wherein dT represents adeoxythimidine nucleotide and sdT represents a deoxythimidine nucleotidehaving a 5′ phosphorothioate.

In another preferred embodiment, an AAT RNAi trigger molecule describedherein comprises a sense strand with a 3′ extension of 1-5 nucleotidesin length, preferably 1-2 nucleotides in length. In one embodiment, oneor more of the antisense strand extension nucleotides comprisesadenosine, uracil, or thymidine nucleotides, AT dinucleotide, ornucleotides which correspond to nucleotides in the AAT mRNA sequence. Ina preferred embodiment, the 3′ sense strand extension consists ofAf(invdT), wherein Af represents a 2′-fluoro Adenosine nucleotide andinvdT represents an inverted (3′-3′-linked) deoxythimidine nucleotide.

In one embodiment, an AAT RNAi trigger molecule described hereincomprises an antisense strand with a 5′ extension of 1-5 nucleotides inlength, preferably 1-2 nucleotides in length. In one embodiment, one ormore of the antisense strand extension nucleotides comprises uracil orthymidine nucleotides or nucleotides which are complementary to thecorresponding AAT mRNA sequence. In a preferred embodiment, theantisense strand extension consists of dT.

In another preferred embodiment, an AAT RNAi trigger molecule describedherein comprises a sense strand with a 5′ extension of 1-5 nucleotidesin length, preferably 1-3 nucleotides in length. In one embodiment, oneor more of the sense strand extension nucleotides comprise uracil oradenosine nucleotides or nucleotides which correspond to nucleotides inthe AAT mRNA sequence. In a preferred embodiment, the sense strandextension consists of 5′ UAU or 5′ uAu, wherein u represents a 2′O-methyl-modified uridine nucleotide.

RNAi trigger molecules described herein may contain 3′ and/or 5′extensions independently on each of the sense strands and antisensestrands. In one embodiment, both the sense strands and the antisensestrands contain 3′ and 5′ extensions, each as described above. In oneembodiment, one or more of the 3′ extension nucleotides of one strandbase pairs with one or more 5′ extension nucleotides of the otherstrand. In another embodiment, the one or more of the 3′ extensionnucleotides of one strand do not base pair with the one or more 5′extension nucleotides of the other strand. The sense and antisensestrands of an RNAi trigger may or may not contain the same number ofnucleotide bases. The antisense and sense strands may form a duplexwherein the 5′ end only has a blunt end, the 3′ end only has a bluntend, both the 5′ and 3′ ends are blunt ended, or neither the 5′ end northe 3′ end are blunt ended. In another embodiment, one or more of thenucleotides in the extension contains a thiophosphate, phosphorothioate,deoxynucleotide inverted (3′ to 3′ linked) nucleotide or is a modifiedribonucleotide or deoxynucleotide.

In some embodiments the sense and antisense strands of the hereindescribed RNAi triggers contain different numbers of nucleotide bases.In some embodiments, the sense strand 5′ end and the antisense strand 3′end of a herein described RNAi trigger form a blunt end. In someembodiments, the sense strand 3′ end and the antisense strand 5′ end ofa herein described RNAi trigger form a blunt end. In some embodiments,the both ends of a herein described RNAi trigger form a blunt end. Insome embodiments, neither end of a herein described RNAi trigger isblunt ended. As used herein a blunt end refers to an end of a doublestranded trigger molecule in which the terminal nucleotides of the twoannealed strands are complementary (form a complementary base-pair). Insome embodiments, the sense strand 5′ end and the antisense strand 3′end of a herein described RNAi trigger form a frayed end. In someembodiments, the sense strand 3′ end and the antisense strand 5′ end ofa herein described RNAi trigger form a frayed end. In some embodiments,the both ends of a herein described RNAi trigger form a frayed end. Insome embodiments, neither end of a herein described RNAi trigger is afrayed end. As used herein a frayed end refers to an end of a doublestranded trigger molecule in which the terminal nucleotides of the twoannealed strands are not complementary (i.e. form a non-complementarybase-pair). As used herein, an overhang is a stretch of one or moreunpaired nucleotides at the end of one strand of a double strand RNAitrigger molecule. The unpaired nucleotides may be on the sense strand orthe antisense strand, creating either 3′ or 5′ overhangs. In someembodiments the RNAi trigger molecule contains: a blunt end and a frayedend, a blunt end and 5′ overhang end, a blunt end and a 3′ overhang end,a frayed end and a 5′ overhand end, a frayed end and a 3′ overhang end,two 5′ overhang ends, two 3′ overhang ends, or a 5′ overhang end and a3′ overhand end.

In one preferred embodiment the inventive AAT RNAi trigger moleculescomprise sequence pairs selected from the group consisting of SEQ IDNOs: 15/23, 15/24, 16/28, 17/30, 18/31, 19/36, and 19/38. In anotherpreferred embodiment the inventive AAT mero RNAi trigger moleculescomprise meroduplexes selected from the group consisting of SEQ ID NOs:15/25/41, 15/26/42, 15/27/43, 16/29/44, 18/32/45, 18/33/46, 18/34/47,18/35/48, and 19/37/49.

RNAi triggers (also called dsRNAi triggers) inhibit gene expressionthrough the biological process of RNA interference (RNAi). RNAi triggerscomprise double stranded RNA or RNA-like structures typically containing15-50 base pairs and preferably 18-25 base pairs and having a nucleobasesequence identical (perfectly complementary) or nearly identical(substantially complementary) to a coding sequence in an expressedtarget gene within the cell. RNAi triggers include, but are not limitedto: short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), microRNAs (miRNAs), short hairpin RNAs (shRNA), meroduplexes, unlockednucleic acid-containing dsRNAs, and dicer substrates (U.S. Pat. No.8,084,599 8,349,809 and 8,513,207).

The AAT RNAi trigger molecules described herein may be comprised ofnaturally occurring nucleotides or may be comprised of at least onemodified nucleotide or nucleotide mimic. The RNAi trigger sense andantisense strands described herein may be synthesized and/or modified bymethods well established in the art.

A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purinecompound which is a constituent of all nucleic acids and includesadenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Anucleoside is a ribosyl or deoxyribosyl derivative of certain pyrimidineor purine bases. They are thus glycosylamines or N-glycosides related tonucleotides by the lack of phosphorylation. It has also become customaryto include among nucleosides analogous substances in which the glycosylgroup is attached to carbon rather than nitrogen (‘C-nucleosides’). Anucleotide is a compound formally obtained by esterification of the 3′or 5′ hydroxy group of nucleosides with phosphoric acid. They are themonomers of nucleic acids.

As used herein, “G,” “C,” “A”, “U” and “T” or “dT” respectively, eachgenerally stand for a nucleobase, nucleoside, nucleotide or nucleotidemimic that contains guanine, cytosine, adenine, uracil anddeoxythymidine as a base, respectively. Also, as used herein, the term“ribonucleotide” or “nucleotide” can also refer to a modified nucleotideor nucleotide mimic, as further detailed below, or a surrogatereplacement moiety. Sequences comprising such replacement moieties areembodiments described herein.

For RNAi trigger molecules described herein, the nucleosides, ornucleotide bases, may be linked by phosphate-containing (natural) ornon-phosphate-containing (non-natural) covalent internucleosidelinkages, i.e. the RNAi trigger molecules may have natural ornon-natural oligonucleotide backbones. In another embodiment, the RNAitrigger contains a non-standard (non-phosphate) linkage between twonucleotide bases.

In a preferred embodiment, one of more nucleotides of the RNAi triggermolecules are modified nucleotides. In another embodiment, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or100% of the nucleotides are modified. Modified nucleotides include, butare not limited to: 2′ modifications, 2′-O-methyl nucleotide(represented herein as a lower case letter ‘n’ in a nucleotidesequence), 2′-deoxy-2′-fluoro nucleotide (represented herein as Nf, alsorepresented herein as 2′ fluoro nucleotide), 2′-deoxy nucleotide(represented herein as dN), 2′-amino nucleotide, 2′-alkyl nucleotide,terminal 3′ to 3′ linkages, inverted deoxythymidine (represented hereinas invdT), a nucleotide comprising a 5′-phosphorothioate group(represented herein as a lower case ‘s’ before a nucleotide, as in sN),thiophosphate linkages, phosphorodithioate group, non-natural basecomprising nucleotide, locked nucleotides, bridged nucleotides, peptidenucleic acids, 2′,3′-seco nucleotide mimic (unlocked nucleotide,represented herein as N_(UNA)), morpholino nucleotides, and abasicnucleotide. It is not necessary for all positions in a given compound tobe uniformly modified. Conversely, more than one modification may beincorporated in a single RNAi trigger compound or even in a singlenucleotide thereof. Ribose 2′ modification may be combined with modifiednucleoside linkages.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl inter-sugarlinkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages,or one or more short chain heteroatomic or heterocyclic inter-sugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

In another embodiment, the AAT RNAi trigger molecules described hereinare canonical siRNAs having modified nucleotides. Exemplary sequencessuitable for forming AAT canonical siRNA RNAi triggers having modifiednucleotides are shown in Table 3. Exemplary AAT canonical siRNA RNAitriggers are SEQ ID pairs: 50/62, 50/63, 53/67, 54/69, 55/70, 56/75, and56/77.

In another embodiment, the AAT RNAi trigger molecules described hereinare meroduplexes having modified nucleotides. Exemplary sequencessuitable for forming AAT meroduplex RNAi triggers having modifiednucleotides are shown in Table 4. Exemplary AAT meroduplex RNAi triggersare SEQ ID meroplexes: 50/64/81, 50/65/82, 50/66/83, 53/68/84, 55/71/85,55/72/86, 55/73/87, 55/74/88, and 56/76/89.

The AAT RNAi trigger antisense strand preferably contains at least one“unlocked nucleotide” (UNA), AAT UNA RNAi trigger. UNA is an acyclic-RNAmimic also known as 2′,3′-seco-RNA, wherein the C2′-C3′ ribose bond isabsent. Because the ribose 2′,3′ bond is absent, UNAs are flexible,enabling modulation of affinity and specificity. UNA exhibit decreasedbinding affinity towards a complementary strand resulting in a decreasein the thermostability of the duplex. A UNA may be located anywherealong a base strand of an RNAi trigger. RNAi triggers described hereinpreferably contain a UNA located at position 6 or 7 (numbering includedthe 5′ dT nucleotide extension). Exemplary sequences suitable forforming AAT UNA RNAi triggers are shown in Table 5. Exemplary AAT UNARNAi triggers are SEQ ID pairs: 51/63, 52/63, 57/77, and 58/77.

Modified nucleobases include other synthetic and natural nucleobases,such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

In one embodiment, AAT RNAi triggers described herein comprise atargeting moiety conjugated to the RNAi trigger. We have found thatconjugation of an RNAi trigger to a targeting moiety, wherein thetargeting moiety comprises a hydrophobic group or to a galactosecluster, facilitates in vivo targeting of the RNAi trigger to the liver.A RNAi trigger-targeting moiety conjugate is formed by covalentlylinking the RNAi trigger to the targeting moiety. The targeting moietymay be linked to the 3′ or the 5′ end of the RNAi trigger sense strandor antisense strand. The targeting moiety is preferably linked to theRNAi trigger sense strand 5′ end.

In one embodiment, the targeting moiety consists of a hydrophobic group.More specifically, the RNAi trigger targeting moiety consists of ahydrophobic group having at least 20 carbon atoms. Hydrophobic groupsused as targeting moieties are herein referred to as hydrophobictargeting moieties. Hydrophobic targeting moieties are preferablyhydrocarbons, containing only carbon and hydrogen atoms. However,substitutions or heteroatoms which maintain hydrophobicity, for examplefluorine, may be permitted. Hydrophobic groups useful as targetingmoieties may be selected from the group consisting of: alkyl group,alkenyl group, alkynyl group, aryl group, aralkyl group, aralkenylgroup, and aralkynyl group, each of which may be linear, branched, orcyclic, cholesterol, cholesterol derivative, sterol, steroid, andsteroid derivative. Exemplary suitable hydrophobic groups may beselected from the group comprising: cholesterol, cholesterolderivatives, dicholesterol, tocopherol, ditocopherol, didecyl,didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoid, andcholeamide.

In another embodiment, the targeting moiety comprises a galactosetargeting moiety or galactose cluster targeting moiety. As used herein,a galactose cluster comprises a molecule having two to four, oftenthree, terminal galactose derivatives. As used herein, the termgalactose derivative includes both galactose and derivatives ofgalactose having affinity for the asialoglycoprotein receptor equal toor greater than that of galactose. Galactose or galactose clustersuseful for targeting oligonucleotides and other molecules to the liverin vivo are well known in the art.

Other terms common in the art include tri-antennary galactose,tri-valent galactose and galactose trimer. A preferred galactosederivative is an N-acetyl-galactosamine (GalNAc). Other saccharideshaving affinity for the asialoglycoprotein receptor may be selected fromthe list comprising: galactose, galactosamine, N-formylgalactosamine,N-acetylgalactosamine, N-propionyl-galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactos-amine.

In one embodiment a targeting moiety is conjugated to the 5′ end of anAAT RNAi trigger sense strand. In another preferred embodiment, thetargeting moiety is conjugated to the 5′ end of an AAT RNAi triggersense strand having a UAU extension. A preferred targeting moiety is acholesteryl derivative. A preferred UAU extension is a uAu extension. Inyet another embodiment, the cholesteryl derivative is linked to the 5′end of the AAT RNAi trigger sense strand via a linker. Exemplary linkersinclude alkyl groups and PEG groups. A preferred PEG linker is atriethylene glycol linkage.

Exemplary AAT RNAi triggers having cholesteryl targeting moietiesinclude: SEQ ID pairs or meroduplexes: 50/63, 56/77, 50/64/81, 50/65/82,50/66/83, 53/68/84, 55/71/85, 55/72/86, 55/73/87, 55/74/88, 56/76/89,51/63, 52/63, 57/77, and 58/77.

The RNAi trigger molecules described herein may be synthesized having areactive group, such as an amine group, at the 5′-terminus. The reactivegroup may be used to subsequently attach a targeting moiety usingmethods typical in the art.

As used herein, the term “sequence” refers to a chain of nucleotidesthat is described by the sequence referred to using the standardnucleotide nomenclature. However, as detailed herein, such a “strandcomprising a sequence” may also comprise modifications, like modifiednucleotides and nucleotide mimics.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence (e.g.RNAi trigger sense strand or AAT mRNA) in relation to a secondnucleotide sequence (e.g. RNAi trigger antisense strand), refers to theability of an oligonucleotide or polynucleotide comprising the firstnucleotide sequence to hybridize and form a duplex or double helicalstructure under certain conditions with an oligonucleotide orpolynucleotide comprising the second nucleotide sequence. Complementarysequences include Watson-Crick base pairs or non-Watson-Crick base pairsand include natural or modified nucleotides or nucleotide mimics in asfar as the above requirements with respect to their ability to hybridizeare fulfilled. Perfectly or fully complementary means that all the basesin a contiguous sequence of a first polynucleotide will hybridize withthe same number of bases in a contiguous sequence of a secondpolynucleotide. The contiguous sequence may comprise all or a part ofthe first or second nucleotide sequence. Partial complementary meansthat in a hybridized pair of nucleobase sequences there are one or moremismatched base pairs. Substantial complementary, as used here whenreferring to the RNAi triggers described herein means that in ahybridized pair of nucleobase sequences 1-3 mismatched base pair. Theterms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of an RNAi trigger, orbetween the antisense strand of a RNAi trigger and a sequence of the AATmRNA.

We describe compositions and methods for inhibiting expression of AAT ina cell, group of cells, tissue, or subject, comprising: administering tothe subject a therapeutically effective amount of a herein described AATRNAi trigger thereby inhibiting the expression of AAT in the subject.Silence, reduce, inhibit, down-regulate, or knockdown gene expression,in as far as they refer to an AAT gene, means that the expression of thegene, as measured by the level of RNA transcribed from the gene or thelevel of polypeptide, protein or protein subunit translated from themRNA in a cell, group of cells, tissue, or subject in which the AAT geneis transcribed, is reduced when the cell, group of cells, tissue, orsubject is treated with AAT RNAi triggers described herein as comparedto a second cell, group of cells, tissue, or subject but which has orhave not been so treated.

Furthermore, the invention relates to a method for inhibiting expressionof the AAT gene in a cell, tissue or organism comprising the steps of:introducing into the cell, tissue or organism an RNAi trigger as definedherein; and maintaining said cell, tissue or organism for a timesufficient to obtain degradation of the mRNA transcript of AAT, therebyinhibiting expression of AAT in a given cell.

In some embodiments, we describe pharmaceutical compositions comprisingat least one of the described AAT RNAi triggers. These pharmaceuticalcompositions are particularly useful in the inhibition of the expressionof the AAT gene in a cell, a tissue, or an organism. The describedpharmaceutical compositions can be used to treat a subject having adisease or disorder that would benefit from reduction or inhibition inAAT expression. The described pharmaceutical compositions can be used totreat a subject at risk of developing a disease or disorder that wouldbenefit from reduction or inhibition in AAT expression. Diseases and/ordisorder that would benefit from reduction or inhibition in AATexpression may be selected from the list comprising: AATD, chronichepatitis, cirrhosis, hepatocellular carcinoma, and fulminant hepaticfailure. Preferably, the subject is a mammal, most preferably a humanpatient. In one embodiment, the method comprises administering acomposition comprising an AAT RNAi trigger molecule described herein toa mammal to be treated. The pharmaceutical compositions described abovemay also comprise a one or more pharmaceutically acceptable excipient(including vehicles, carriers, diluents, and/or delivery polymers).

In another embodiment, the invention provides methods for treating,preventing or managing clinical presentations associated with AATDincluding, AATD. Said methods comprise administering to a subject inneed of such treatment, prevention or management a therapeutically orprophylactically effective amount of one or more of the AAT RNAitriggers described herein. Preferably, said subject is a mammal, mostpreferably a human patient. In one embodiment, the method comprisesadministering a composition comprising an AAT RNAi trigger moleculedescribed herein to a mammal to be treated.

The terms “treat”, “treatment”, and the like, mean in context of thisinvention the relief from or alleviation of a disorder related to AATD.

The described AAT RNAi triggers and methods can be used to treat orprevent at least one symptom in a subject having a disease or disorderthat would benefit from reduction or inhibition in AAT expression. Thesubject is administered a therapeutically effective amount of any one ormore of the described RNAi triggers thereby treating the symptom. Thesubject is administered a prophylactically effective amount of any oneor more of the described RNAi triggers thereby preventing the at leastone symptom.

In some embodiments, the gene expression level and/or mRNA level of AATin a subject to whom a described AAT RNAi trigger is administered isreduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to thesubject not receiving the AAT RNAi trigger. The gene expression leveland/or mRNA level in the subject may be reduced in a cell, group ofcells, and/or tissue of the subject. In some embodiments, the proteinlevel of AAT in a subject to whom a described AAT RNAi trigger isadministered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%relative to the subject not receiving the AAT RNAi trigger. The proteinlevel in the subject may be reduced in a cell, group of cells, tissue,blood, and/or other fluid of the subject. Reduction in gene expression,mRNA, or protein levels can be assessed by any methods known in the art.Reduction or decrease in AAT mRNA level and/or protein level arecollectively referred to herein as a reduction or decrease in AAT orinhibiting or reducing the expression of AAT.

“Introducing into a cell”, when referring to a RNAi trigger, meansfunctionally delivering the RNAi trigger into a cell. By functionaldelivery, it is meant that the RNAi trigger is delivered to the cell andhas the expected biological activity, sequence-specific inhibition ofgene expression. Many molecules, including RNAi trigger molecules,administered to the vasculature of a mammal are normally cleared fromthe body by the liver. Clearance of a an RNAi trigger by the liverwherein the RNAi trigger is degraded or otherwise processed for removalfrom the body and wherein the RNAi trigger does not causesequence-specific inhibition of gene expression is not consideredfunctional delivery.

The route of administration is the path by which a RNAi trigger isbrought into contact with the body. In general, methods of administeringdrugs and nucleic acids for treatment of a mammal are well known in theart and can be applied to administration of the compositions describedherein. The compounds of the present invention can be administered viaany suitable route in a preparation appropriately tailored to theparticular route. Thus, the compounds of the present invention can beadministered by injection, for example, intravenously, intramuscularly,intracutaneously, subcutaneously, or intraperitoneally. Accordingly, thepresent invention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier or excipient.

The AAT RNAi trigger molecules or compositions described herein can bedelivered to a cell, group of cells, tissue, or subject usingoligonucleotide delivery technologies known in the art. In general, anysuitable method recognized in the art for delivering a nucleic acidmolecule (in vitro or in vivo) can be adapted for use with an RNAitrigger of the invention. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal),intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

The RNAi triggers can be combined with lipids, nanoparticles, polymers,liposomes, micelles, DPCs or other delivery systems available in theart. The RNAi triggers can also be chemically conjugated to targetingmoieties, lipids (including, but not limited to cholesterol andcholesteryl derivative), nanoparticles, polymers, liposomes, micelles,DPCs (WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO2012/083185, each of which is incorporated herein by reference), orother delivery systems available in the art.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of at least one kind of RNAi triggerand a pharmaceutically acceptable carrier and optionally an excipientone or more a pharmaceutically acceptable excipients. Pharmaceuticallyacceptable excipients (excipients) are substances other than the ActivePharmaceutical ingredient (API, therapeutic product, e.g., AAT RNAitrigger) that have been appropriately evaluated for safety and areintentionally included in the drug delivery system. Excipients do notexert or are not intended to exert a therapeutic effect at the intendeddosage. Excipients may act to a) aid in processing of the drug deliverysystem during manufacture, b) protect, support or enhance stability,bioavailability or patient acceptability of the API, c) assist inproduct identification, and/or d) enhance any other attribute of theoverall safety, effectiveness, of delivery of the API during storage oruse.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, binders,buffering agents, carriers, coating agents, colors, delivery enhancers,dextran, dextrose, diluents, disintegrants, emulsifiers, extenders,fillers, flavors, glidants, humectants, lubricants, oils, polymers,preservatives, saline, salts, solvents, sugars, suspending agents,sustained release matices, sweeteners, thickening agents, tonicityagents, vehicles, water-repelling agents, and wetting agents. Apharmaceutically acceptable excipient may or may not be an inertsubstance.

The pharmaceutical compositions of the can contain other additionalcomponents commonly found in pharmaceutical compositions. Thepharmaceutically-active materials may include, but are not limited to:anti-pruritics, astringents, local anesthetics, or anti-inflammatoryagents (e.g., antihistamine, diphenhydramine, etc.). It is alsoenvisaged that cells, tissues or isolated organs that express orcomprise the herein defined RNAi triggers may be used as “pharmaceuticalcompositions”. As used herein, “pharmacologically effective amount,”“therapeutically effective amount,” or simply “effective amount” refersto that amount of an RNAi trigger to produce the intendedpharmacological, therapeutic or preventive result.

In one embodiment, the AAT RNAi trigger-targeting moiety conjugate isco-administered with an MLP delivery polymer (excipient). Byco-administered it is meant that the AAT RNAi trigger and the deliverypolymer are administered to the mammal such that both are present in themammal at the same time. The AAT RNAi trigger-targeting moiety conjugateand the delivery polymer may be administered simultaneously or they maybe delivered sequentially. For simultaneous administration, they may bemixed prior to administration. For sequential administration, either theAAT RNAi trigger-targeting moiety conjugate or the delivery polymer maybe administered first.

In a preferred embodiment, the invention features a pharmaceuticalcomposition for delivering an AAT RNAi trigger to a liver cell in vivocomprising: a) an AAT RNAi trigger conjugated to a hydrophobic groupcontaining at least 20 carbon atoms (RNA trigger-conjugate), such as acholesterol and b) an MLP delivery polymer. The MLP delivery polymer andthe RNA trigger-conjugate are synthesized separately and may be suppliedin separate containers or a single container. In a preferred embodiment,the AAT RNAi trigger is not conjugated to the delivery polymer.

MLP Delivery Polymer

Melittin-like peptide, MLP, as used herein, is a small amphipathicmembrane active peptide, comprising about 23 to about 32 amino acidsderived from the naturally occurring bee venom peptide, melittin, asdescribed in WO 2012/083185. The naturally occurring melittin contains26 amino acids and is predominantly hydrophobic on the amino terminalend and predominantly hydrophilic (cationic) on the carboxy terminalend. MLP described herein can be isolated from a biological source or itcan be synthetic. A synthetic polymer is formulated or manufactured by achemical process “by man” and is not created by a naturally occurringbiological process. As used herein, MLP encompasses the naturallyoccurring bee venom peptides of the melittin family that can be foundin, for example, venom of the species: Apis florea, Apis mellifera, Apiscerana, Apis dorsata, Vespula maculifrons, Vespa magnifica, Vespavelutina, Polistes sp. HQL-2001, and Polistes hebraeus. As used herein,MLP also encompasses synthetic peptides having amino acid sequenceidentical to or similar to naturally occurring melittin peptides.Exemplary MLP amino acid sequences include those shown in FIG. 6. Inaddition to the amino acids which retain melittin's inherent highmembrane activity, 1-8 amino acids can be added to the amino or carboxyterminal ends of the peptide. Specifically, cysteine residues can beadded to the amino or carboxy termini. The list in FIG. 1 is not meantto be exhaustive, as other conservative amino acid substitutions arereadily envisioned. Synthetic MLPs can contain naturally occurring Lform amino acids or the enantiomeric D form amino acids (inverso).However, a MLP should either contain essentially all L form or all Dform amino acids but may have amino acids of the opposite stereocenterappended at either the amino or carboxy termini. The MLP amino acidsequence can also be reversed (retro). Retro MLP can have L form aminoacids or D form amino acids (retroinverso). MLPs can have modifyinggroups, other than masking agents, that enhance tissue targeting orfacilitate in vivo circulation attached to either the amino terminal orcarboxy terminal ends of the peptide. However, as used herein, MLP doesnot include chains or polymers containing more than two MLP peptidescovalently linked to one another other or to another polymer orscaffold.

In one embodiment, a MLP comprises an Apis florea (little or dwarf honeybee) melittin, Apis mellifera (western or European or big honey bee),Apis dorsata (giant honey bee), Apis cerana (oriental honey bee) orderivatives thereof. A preferred MLP comprises the sequence:Xaa₁-Xaa₂-Xaa₃-Ala-Xaa₅-Leu-Xaa₇-Val-Leu-Xaa₁₀-Xaa₁₁-Xaa₁₂-Leu-Pro-Xaa₁₅-Leu-Xaa₁₇-Xaa₁₈-Trp-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆wherein:

-   -   Xaa₁ is leucine, D-leucine, isoleucine, norleucine, tyrosine,        tryptophan, valine, alanine, dimethylglycine, glycine,        histidine, phenylalanine, or cysteine,    -   Xaa₂ is isoleucine, leucine, norleucine, or valine,    -   Xaa₃ is glycine, leucine, or valine,    -   Xaa₅ is isoleucine, leucine, norleucine, or valine,    -   Xaa₇ is lysine, serine, asparagine, alanine, arginine, or        histidine,    -   Xaa₁₀ is alanine, threonine, or leucine,    -   Xaa₁₁ is threonine or cysteine,    -   Xaa₁₂ is glycine, leucine, or tryptophan,    -   Xaa₁₅ is threonine or alanine,    -   Xaa₁₇ is isoleucine, leucine, norleucine, or valine,    -   Xaa₁₈ is serine or cysteine,    -   Xaa₂₀ is isoleucine, leucine, norleucine, or valine,    -   Xaa₂₁ is lysine or alanine,    -   Xaa₂₂ is asparagine or arginine,    -   Xaa₂₃ is lysine or alanine,    -   Xaa₂₄ is arginine or lysine,    -   Xaa₂₅ is lysine, alanine, or glutamine,    -   Xaa₂₆ is optional and if present is glutamine, cysteine,        glutamine-NH₂, or cysteine-NH₂; and, and at least two of Xaa₂₁,        Xaa₂₃, and Xaa₂₅ are lysine.

In another embodiment, MLP comprises the sequence:Xaa₁-Xaa₂-Xaa₃-Ala-Xaa₅-Leu-Xaa₇-Val-Leu-Xaa₁₀-Xaa₁₁-Xaa₁₂-Leu-Pro-Xaa₁₅-Leu-Xaa₁₇-Ser-Trp-Xaa₂₀-Lys-Xaa₂₂-Lys-Arg-Lys-Xaa₂₆wherein:

-   -   Xaa₁ is leucine, D-leucine, norleucine, or tyrosine,    -   Xaa₂ is isoleucine, leucine, norleucine, or valine,    -   Xaa₃ is glycine, leucine, or valine,    -   Xaa₅ is isoleucine, valine, leucine, or norleucine,    -   Xaa₇ is lysine, serine, asparagine, alanine, arginine, or        histidine,    -   Xaa₁₀ is alanine, threonine, or leucine,    -   Xaa₁₁ is threonine, or cysteine,    -   Xaa₁₂ is glycine, leucine, or tryptophan,    -   Xaa₁₅ is threonine, or alanine,    -   Xaa₁₇ is isoleucine, leucine, or norleucine,    -   Xaa₂₀ is isoleucine, leucine, or norleucine,    -   Xaa₂₂ is asparagine or arginine, and    -   Xaa₂₆ is glutamine or cysteine.

A another embodiment, MLP comprises the sequence:Xaa₁-Xaa₂-Gly-Ala-Xaa₅-Leu-Lys-Val-Leu-Ala-Xaa₁₁-Gly-Leu-Pro-Thr-Leu-Xaa₁₇-Ser-Trp-Xaa₂₀-Lys-Xaa₂₂-Lys-Arg-Lys-Xaa₂₆wherein:

-   -   Xaa₁, Xaa₂, Xaa₅, Xaa₁₇ and Xaa₂₀ are independently isoleucine,        leucine, or norleucine,    -   Xaa₁₁ is threonine or cysteine,    -   Xaa₂₂ is Asparagine or arginine, and    -   Xaa₂₆ is glutamine or cysteine.

A another embodiment, MLP comprises:Leu-Ile-Gly-Ala-Ile-Leu-Lys-Val-Leu-Ale-Thr-Gly-Leu-Pro-Thr-Leu-Ile-Ser-Trp-Ile-Lys-Asn-Lys-Arg-Lys-Gln.

MLPs described herein are membrane active and therefore capable ofdisrupting plasma membranes or lysosomal/endocytic membranes. As usedherein, membrane active peptides are surface active, amphipathicpeptides that are able to induce one or more of the following effectsupon a biological membrane: an alteration or disruption of the membranethat allows non-membrane permeable molecules to enter a cell or crossthe membrane, pore formation in the membrane, fission of membranes, ordisruption or dissolving of the membrane. As used herein, a membrane, orcell membrane, comprises a lipid bilayer. The alteration or disruptionof the membrane can be functionally defined by the peptide's activity inat least one the following assays: red blood cell lysis (hemolysis),liposome leakage, liposome fusion, cell fusion, cell lysis, andendosomal release. Peptides that preferentially cause disruption ofendosomes or lysosomes over plasma membranes are consideredendosomolytic. The effect of membrane active peptides on a cell membranemay be transient. Membrane active peptides possess affinity for themembrane and cause a denaturation or deformation of bilayer structures.Delivery of a RNAi trigger to a cell is mediated by the MLP disruptingor destabilizing the plasma membrane or an internal vesicle membrane(such as an endosome or lysosome), including forming a pore in themembrane, or disrupting endosomal or lysosomal vesicles therebypermitting release of the contents of the vesicle into the cellcytoplasm.

Membrane activity of the MLPs is reversibly masked to yield MLP deliverypolymers. Masking is accomplished through reversible attachment ofmasking agents to primary amines of the MLP.

It is an essential feature of the masking agents that, in aggregate,they inhibit membrane activity of the MLP and provide in vivo hepatocytetargeting. As used herein, MLP is masked if the modified MLP (MLPdelivery polymer) does not exhibit membrane activity and exhibitscell-specific (i.e. hepatocyte) targeting in vivo. MLP is reversiblymasked if cleavage of bonds linking the masking agents to the peptideresults in restoration of amines on the MLP thereby restoring membraneactivity. It is an essential feature that the masking agents arecovalently bound to the MLP through physiologically labile reversiblebonds. By using physiologically labile reversible linkages or bonds, themasking agents can be cleaved from the MLP in vivo, thereby unmaskingthe MLP and restoring activity of the unmasked MLP. A sufficient numberof masking agents are linked to the MLP to achieve the desired level ofinactivation. The desired level of modification of MLP by attachment ofmasking agent(s) is readily determined using appropriate peptideactivity assays. For example, if MLP possesses membrane activity in agiven assay, a sufficient level of masking agent is linked to the MLP toachieve the desired level of inhibition of membrane activity in thatassay. Modification of ≥80% or ≥90% of the primary amine groups on apopulation of MLP peptides, as determined by the quantity of primaryamines on the peptides in the absence of any masking agents, ispreferred. It is also a preferred characteristic of masking agents thattheir attachment to the peptide reduces positive charge of the polymer,thus forming a more neutral delivery polymer. It is desirable that themasked peptide retain aqueous solubility.

An MLP delivery polymer comprises an MLP reversibly modified by reactionof primary amines on the peptide with asialoglycoprotein receptor(ASGPr) ligand-containing masking agents wherein said reversiblymodification is physiologically labile, as described in WO 2012/083185.

As used herein, a masking agent comprises a preferably neutral(uncharged) compound having an ASGPr ligand and an amine-reactive groupwherein reaction of the amine-reactive group with an amine on a peptideresults in linkage of the ASGPr ligand to the peptide via a reversiblephysiologically labile covalent bond. An amine is reversibly modified ifcleavage of the modifying group restores the amine. A preferred ASGPrligand-containing masking agent has a neutral charge and comprises aASGPr ligand, such as a galactosamine or galactosamine derivative,having a disubstituted maleic anhydride amine-reactive group. Themembrane active polyamine can be conjugated to masking agents in thepresence of an excess of masking agents. The excess masking agent may beremoved from the conjugated delivery polymer prior to administration ofthe delivery polymer.

Galactose and galactose derivatives have been used to target moleculesto hepatocytes in vivo through their binding to the asialoglycoproteinreceptor (ASGPr) expressed on the surface of hepatocytes. As usedherein, a ASGPr ligand (or ASGPr ligand) comprises a galactose andgalactose derivative having affinity for the ASGPr equal to or greaterthan that of galactose. Binding of galactose targeting moieties to theASGPr(s) facilitates cell-specific targeting of the delivery polymer tohepatocytes and endocytosis of the delivery polymer into hepatocytes.ASGPr ligands may be selected from the group comprising: lactose,galactose, N-acetylgalactosamine (GalNAc), galactosamine,N-formylgalactosamine, N-acetyl-galactosamine, N-propionylgalactosamine,N-n-butanoylgalactosamine, and N-iso-butanoyl-galactosamine (Iobst, S.T. and Drickamer, K. J.B.C. 1996, 271, 6686). ASGPr ligands can bemonomeric (e.g., having a single galactosamine) or multimeric (e.g.,having multiple galactosamines).

A preferred masking agent comprises a neutral hydrophilic disubstitutedalkylmaleic anhydride:

wherein R1 comprises an uncharged asialoglycoprotein receptor ligand. Apreferred alkyl group is a methyl or ethyl group. An example of asubstituted alkylmaleic anhydride consists of a2-propionic-3-alkylmaleic anhydride derivative. A neutral hydrophilic2-propionic-3-alkylmaleic anhydride derivative is formed by attachmentof a neutral hydrophilic group to a 2-propionic-3-alkylmaleic anhydridethrough the 2-propionic-3-alkylmaleic anhydride γ-carboxyl group:

wherein R1 comprises a neutral ASGPr ligand and n=0 or 1. In oneembodiment, the ASGPr ligand is linked to the anhydride via a short PEGlinker.

The ASGPr ligand provides targeting function through affinity for ASGPr.Preferred ASGPr ligands contain saccharides having affinity for theASGPr, including but not limited to: galactose, N-acetyl-galactosamineand galactose derivatives. Galactose derivatives having affinity for theASGPr are well known in the art.

The invention includes conjugate delivery systems of the composition:N-T and MLP-(L-M)_(x),wherein N is a AAT RNAi trigger, T comprises a hydrophobic group having20 or more carbon atoms, MLP is a melittin-like peptide as describedherein, and M contains an ASGPr ligand as described herein covalentlylinked to MLP via a physiologically labile reversible maleamate linkageL. Cleavage of L restores an unmodified amine on MLP. x is an integergreater than 1. More specifically, the value of x is greater than 80%and up to 100% of the number of primary amines on a population MLP. Asused herein, MLP-(L-M)_(x) is an MLP delivery polymer.

Sufficient percentage of MLP primary amines are modified to inhibitmembrane activity of the peptide and provide for hepatocyte targeting.Preferably x has a value greater than 80%, and more preferably greaterthan 90%, of the number of primary amines on a population of MLP, asdetermined by the quantity of amines on the population of MLP in theabsence of any masking agents. It is noted that a single MLP typicallycontains 3-5 primary amines (the amino terminus (if unmodified) andtypically 2-4 Lysine residues). In its unmodified state, MLP is membraneactive. However, MLP delivery polymer, MLP-(L-M)_(x), is not membraneactive. Reversible modification of MLP primary amines, by attachment ofM, reversibly inhibits or inactivates membrane activity of MLP. Uponcleavage of reversible linkages L, unmodified amines are restoredthereby reverting the MLP to its unmodified, membrane active state.MLP-(L-M)_(x), an ASGPr-targeted reversibly masked membrane activepolymer, and T-N, an RNAi trigger-conjugate, are synthesized ormanufactured separately. Neither T nor N are covalently linked directlyor indirectly to MLP, L, or M. Electrostatic or hydrophobic associationof the RNAi trigger or the RNAi-trigger-conjugate with the masked orunmasked polymer is not required for in vivo liver delivery of theRNAi-trigger. The masked polymer and the RNAi-trigger conjugate can besupplied in the same container or in separate containers. They may becombined prior to administration, co-administered, or administeredsequentially.

In one aspect, we describe a pharmaceutical composition for inhibitingexpression of a AAT gene comprising a described herein AAT RNAi triggerdescribed herein.

In one embodiment, the RNAi trigger is administered in an unbufferedsolution. In one embodiment, the unbuffered solution is saline or water.In one embodiment, the RNAi trigger is administered with a buffersolution. In one embodiment, the buffer solution comprises acetate,citrate, prolamine, carbonate, or phosphate or any combination thereof.In another embodiment, the buffer solution is phosphate buffered saline(PBS).

Administration of a described AAT RNAi trigger according to the methodsand uses described herein may result in a reduction of the severity,signs, symptoms, and/or markers of such diseases or disorders in asubject having a disorder that would benefit from inhibiting or reducingthe expression of AAT, such as AATD. Efficacy of treatment or preventionof disease can be assessed, for example by measuring diseaseprogression, disease remission, symptom severity, reduction in pain,quality of life, dose of a medication required to sustain a treatmenteffect, level of a disease marker or any other measurable parameterappropriate for a given disease being treated or targeted forprevention.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an AAT RNAi trigger or co-treatment, that, whenadministered to a subject having a AATD, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the AATRNAi trigger, co-treatment, how the trigger is administered, the diseaseand its severity and the history, age, weight, family history, geneticmakeup, the types of preceding or concomitant treatments, if any, andother individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of a AAT RNAi trigger agent or co-treatments, that,when administered to a subject having a AATD but not yet (or currently)experiencing or displaying symptoms of the disease, and/or a subject atrisk of developing a AATD, is sufficient to prevent or ameliorate thedisease or one or more symptoms of the disease. Ameliorating the diseaseincludes slowing the course of the disease or reducing the severity oflater-developing disease.

In one embodiment, the dose can be: 0.0005, 0.001, 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/kg.Values intermediate to the recited values are also intended to be partof this invention.

In another embodiment, the dose can be: 0.1 to 50, 0.25 to 50, 0.5 to50, 0.75 to 50, 1 to 50, 1.5 to 50, 2 to 50, 2.5 to 50, 3 to 50, 3.5 to50, 4 to 50, 4.5 to 50, 5 to 50, 7.5 to 50, 10 to 50, 15 to 50, 20 to50, 20 to 50, 25 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to50 mg/kg.

In another embodiment, the dose can be: 0.1 to 45, 0.25 to 45, 0.5 to45, 0.75 to 45, 1 to 45, 1.5 to 45, 2 to 45, 2.5 to 45, 3 to 45, 3.5 to45, 4 to 45, 4.5 to 45, 5 to 45, 7.5 to 45, 10 to 45, 15 to 45, 20 to45, 20 to 45, 25 to 45, 25 to 45, 30 to 45, 35 to 45, or 40 to 45 mg/kg.

In another embodiment, the dose can be: 0.1 to 40, 0.25 to 40, 0.5 to40, 0.75 to 40, 1 to 40, 1.5 to 40, 2 to 40, 2.5 to 40, 3 to 40, 3.5 to40, 4 to 40, 4.5 to 40, 5 to 40, 7.5 to 40, 10 to 40, 15 to 40, 20 to40, 20 to 40, 25 to 40, 25 to 40, 30 to 40, or 35 to 40 mg/kg.

In another embodiment, the dose can be: 0.1 to 30, 0.25 to 30, 0.5 to30, 0.75 to 30, 1 to 30, 1.5 to 30, 2 to 30, 2.5 to 30, 3 to 30, 3.5 to30, 4 to 30, 4.5 to 30, 5 to 30, 7.5 to 30, 10 to 30, 15 to 30, 20 to30, 20 to 30, 25 to 30 mg/kg.

In another embodiment, the dose can be: 0.1 to 20, 0.25 to 20, 0.5 to20, 0.75 to 20, 1 to 20, 1.5 to 20, 2 to 20, 2.5 to 20, 3 to 20, 3.5 to20, 4 to 20, 4.5 to 20, 5 to 20, 7.5 to 20, 10 to 20, or 15 to 20 mg/kg.

In another embodiment, the dose can be: 0.01 to 10, 0.05 to 10, 0.1 to10, 0.2 to 10, 0.3 to 10, 0.4 to 10, 0.5 to 10, 1 to 10, 1.5 to 10, 2 to10, 2.5 to 10, 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10,or 9.5 to 10 mg/kg.

In another embodiment, the dose can be: 0.01 to 5, 0.05 to 5, 0.1 to 5,0.2 to 5, 0.3 to 5, 0.4 to 5, 0.5 to 5, 1 to 5, 1.5 to 5, 2 to 5, 2.5 to5, 3 to 5, 3.5 to 5, 4 to 5, or 4.5 to 5 mg/kg.

In another embodiment, the dose can be: 0.01 to 2.5, 0.05 to 2.5, 0.1 to2.5, 0.2 to 2.5, 0.3 to 2.5, 0.4 to 2.5, 0.5 to 5, 1 to 2.5, 1.5 to 2.5,or 2 to 2.5 mg/kg.

In one embodiment, the AAT RNAi trigger is administered once. In anotherembodiment, administration of the AAT RNAi trigger is repeated (i.e.,repeat-dose regimen or multi-dose regimen). For repeatedadministrations, the AAT RNAi trigger may be administered to the subjectonce per day, every other day, once every three days, once every fourdays, once every five days, once every six days, twice a week, once aweek, once every two weeks, once every three weeks, once every fourweeks, once every four (4) to fourteen (14) weeks, twice a month, once amonth, once every two months, once every three months less, or onceevery four months or longer. Values intermediate to the recited valuesare also intended to be part of this invention. In another embodiment,the AAT trigger can be administered as necessary. In another embodiment,an initial treatment regimen may comprise repeat administration at aninitial time interval and subsequent administration on a less frequentbasis. For example, after administration weekly or biweekly for one tosix months, administration can thereafter be repeated once per month orless. The initial time interval can be a set number of administrations,a set span of time, or until a determined reduction in AAT is measured.For any dosing regimen, whether single or repeat, any of the aboveamounts may be used. For repeat dosing, the same dose or different dosesmay be used for each administration.

The invention also provides for cells comprising at least one of theRNAi triggers described herein. The cell is preferably a mammalian cell,such as a human cell. Furthermore, tissues and/or non-human organismscomprising the herein defined RNAi trigger molecules are an embodimentof this invention, whereby said non-human organisms are particularlyuseful for research purposes or as research tools, for example in drugtesting.

The above provided embodiments and items of the present invention arenow illustrated with the following, non-limiting examples.

EXAMPLES Example 1. Identification of RNAi Trigger Sequences

A selection process for identifying lead UNAs to target AAT began within silico methods to identify conserved sequences across variants of theAAT gene. The AAT cDNA sequence was initially screened for 17-nucleotidesequences having an exact complementary sequence in eleven knownvariants of human AAT. Sequences known to have manufacturing challenges,such as runs of five (5) or more guanines or cytosines, and thosepredicted to have poor RNAi activity based on known siRNA parameterswere eliminated. Sequences that included a single nucleotidepolymorphism (SNP) with a major allele frequency of greater than 0.2 atposition 2 to 18 of a 19-mer sequence were also eliminated. Sequenceswere then subjected to cross-species reactivity analysis to selectcandidates that would cross-react with cynomolgus monkey AAT. In silicoanalysis yielded 840 sequences that were 19-mers and 939 sequences thatwere 17-mers. These sequences were then evaluated for specificity toavoid off-target effects against the human and cynomolgus genomes.Sequences containing a conserved miRNA seed region in positions 2-7 ofeither siRNA strand with off-target genes were eliminated. 47 candidatesequences were then selected for use in generating RNAi triggermolecules.

Example 2. RNAi Trigger Synthesis

A) Synthesis. RNAi trigger molecules were synthesized according tophosphoramidite technology on solid phase used in oligonucleotidesynthesis. Depending on the scale either a MerMade96E (Bioautomation) ora MerMade12 (Bioautomation) was used. Syntheses were performed on asolid support made of controlled pore glass (CPG, 500 Å for dT and 600 Åfor inverse dT, obtained from Prime Synthesis, Aston, Pa., USA). All2′-modified RNA phosphoramidites as well as ancillary reagents werepurchased from Thermo Fisher Scientific (Milwaukee, Wis., USA).Specifically, the following 2′-O-Methyl phosphoramidites were used:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropy-lamino)phosphoramidite,5′-O-dimethoxy-trityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite,and5′-O-dimethoxy-trityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite.The 2′-Deoxy-2′-fluoro-phosphor-amidites carried the same protectinggroups as the 2′-O-methyl RNA amidites.5′-(4,4′-Dimethoxytrityl)-2′,3′-seco-uridine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite waspurchased from Link Technologies Ltd, Scotland. All amidites weredissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å)were added. In order to introduce the TEG-Cholesterol at the 5′-end ofthe oligomers, the1-Dimethoxytrityloxy-3-O—(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramiditefrom Glen Research (Sterling, Va., USA) was employed. The5′-modifications were introduced without any modification of thesynthesis cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile)was used as activator solution. Coupling times were 10 min (RNA), 180sec (Cholesterol), 90 sec (2′OMe and UNA), and 60 sec (2′F and DNA). Inorder to introduce phosphorothioate linkages, a 100 mM solution of3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc.,Leominster, Mass., USA) in anhydrous Acetonitrile was employed. SeeTables 1-5 (FIG. 1-5). For

B. Cleavage and Deprotection of Support Bound Oligomer.

After finalization of the solid phase synthesis, the dried solid supporttreated with a 1:1 volume solution of 40 wt. % methylamine in water and28% ammonium hydroxide solution (Aldrich) for two hours at roomtemperature. The solution was evaporated and the solid residue wasreconstituted in water (see below).

C. Purification.

Crude Cholesterol containing oligomers were purified by reverse phaseHPLC using a Waters XBridge BEH300 C4 5u Prep column and a Shimadzu LC-8system. Buffer A was 100 mM TEAA, pH 7.5 and contained 5% Acetonitrileand buffer B was 100 mM TEAA and contained 95% Acetonitrile. A gradientof 45% B to 55% B over 25 minutes was employed. UV traces at 260 nm wererecorded. Appropriate fractions were then run on size exclusion HPLCusing a GE Healthcare XK 16/40 column packed with Sephadex G-25 mediumwith a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20%Acetonitrile. Other crude oligomers were purified by anionic exchangeHPLC using a TKSgel SuperQ-5PW 13u column and Shimadzu LC-8 system.Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20%Acetonitrile and buffer B was the same as buffer A with the addition of1.5 M sodium chloride. A gradient of 32.5% B to 42.5% B over 25 minuteswas employed. UV traces at 260 nm were recorded. Appropriate fractionswere pooled then run on size exclusion HPLC as described for Cholesterolcontaining oligomers.

D. Annealing.

Complementary strands were mixed by combining equimolar RNA solutions(sense and antisense) in 0.2×PBS (Phosphate-Buffered Saline, 1×,Corning, Cellgro) to form the RNAi triggers. This solution was placedinto a thermomixer at 70° C., heated to 95° C., held at 95° C. for 5min, and cooled to room temperature slowly. Some RNAi triggers werelyophilized and stored at −15 to −25° C. Duplex concentration wasdetermined by measuring the solution absorbance on a UV-Vis spectrometerin 0.2×PBS. The solution absorbance at 260 nm was then multiplied by aconversion factor and the dilution factor to determine the duplexconcentration. Unless otherwise stated, all conversion factor was 0.037mg/(mL·cm). For some experiments, a conversion factor of 0.0502mg/(mL·cm) was used.

Example 3. Melittin-Like-Peptide (MLP) Delivery Polymer

A) Melittin-Like-Peptide (MLP) Synthesis.

All MLPs were made using peptide synthesis techniques standard in theart. Suitable MLPs can be all L-form amino acids, all D-form amino acids(inverso). Independently of L or D form, the MLP sequence can bereversed (retro).

B) CDM-NAG (N-acetyl Galactosamine) Synthesis.

To a solution of CDM (300 mg, 0.16 mmol) in 50 mL methylene chloride wasadded oxalyl chloride (2 g, 10 wt. eq.) and dimethylformamide (5 μl).The reaction was allowed to proceed overnight, after which the excessoxalyl chloride and methylene chloride were removed by rotaryevaporation to yield the CDM acid chloride. The acid chloride wasdissolved in 1 mL of methylene chloride. To this solution was added 1.1molar equivalents(aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-β-D-galactopyranoside (i.e.amino bisethoxyl-ethyl NAG) and pyridine (200 μl, 1.5 eq) in 10 mL ofmethylene chloride. The solution was then stirred 1.5 h. The solvent wasthen removed and the resulting solid was dissolved into 5 mL of waterand purified using reverse-phase HPLC using a 0.1% TFAwater/acetonitrile gradient.

R1 comprises a neutral ASGPr ligand. Preferably the Masking Agent isuncharged.

n is an integer from 1 to 10. As shown above, a PEG spacer may bepositioned between the anhydride group and the ASGPr ligand. A preferredPEG spacer contains 1-10 ethylene units. Alternatively an alkyl spacermay be used between the anhydride and the N-Acetylgalactosamine.

n is a integer from 0 to 6.

Other spacers or linkers may be used between the anhydride and theN-Acetyl-galactosamine. However, a hydrophilic, neutral (preferablyuncharged) spacer or linker is preferred.

C) Formation of the MLP Delivery Polymer (i.e. Masking).

The MLP was reacted with CDM-NAG masking agent to yield the MLP deliverypolymer. The MLP component was first dissolved to a final concentrationof 8.5 mg/mL in aqueous HEPES (sodium salt, GMP grade, ˜430 mg/mL). TheMLP solution was then cooled to 4° C., and checked for appearance (clearto pale yellow solution free of visible particulate) and forconcentration by UV spectrophotometry. CDM-NAG was dissolved in water at4° C. at a final concentration of ˜75 mg/mL. The solution was checkedfor appearance (clear to pale yellow solution free of visibleparticulate) and for concentration by UV spectrophotometry. MLP insolution was mixed with CDM-NAG in solution at a 5:1 (w/w) ratio ofCDM-NAG to MLP. The addition rate of CDM-NAG solution was approximately0.3 L per minute, while stirring. After all CDM-NAG solution had beenadded to the MLP solution, the mixture was stirred for 30 minutes. Tostabilize the MLP delivery polymer, the pH was increased to 9.0±0.2 byaddition of 1 M aqueous sodium hydroxide. Reaction of disubstitutedmaleic anhydride masking agent with the peptide yielded a compoundhaving the structure represented by:

wherein R is MLP and R1 comprises an ASGPr ligand (e.g. NAG).

Colorimetric trinitrobenzene sulfonic acid (TNBS) assay of remainingfree amines was used to determine that MLP was sufficiently masked byCDM-NAG, less than 10% of the total number of MLP amines remainedunmodified.

MLP delivery polymer was purified by diafiltration against 10 mM, pH 9.2carbonate buffer to remove excess CDM-NAG. The diafiltration processexchanged ˜10 volumes of carbonate buffer per volume of masked MLPreaction solution and held at 2-8° C.

Component Quantity (nominal) MLP-EX1 Acetate  30 g/L CDM-NAG^(a)  25 g/LSodium carbonate 0.3 g/L Sodium bicarbonate 0.6 g/L Water 1000 g/L  ^(a)assumes five (5) CDM-NAG moieties per MLP

The MLP delivery polymer was further formulated with Dextran to 10% w/vand stored at 2 to 8° C. For some experiments, this solution waslyophilized prior to use.

D) Injection Solution.

The injection solution was formed by mixing RNAi trigger with the MLPdelivery polymer. The lyophilized MLP delivery polymer was dissolved inwater and mixed with the RNAi trigger. That solution was then diluted tothe correct injection concentration with normal saline.

Example 4. In Vitro Screening of siRNAs

Candidate sequences identified by in silico analysis (Example 1) werescreened as chemically modified canonical siRNAs in vitro. Forty-six ofthe in silico identified potential AAT RNAi triggers were synthesized ascanonical siRNAs and screened for efficacy in vitro. Hep3B cells, ahuman hepatocellular carcinoma line, were plated at ˜10,000 cells perwell in 96-well format. Each of the 46 siRNAs was transfected at twoconcentrations, 1 nM and 10 nM, in triplicate, using LipofectamineRNAiMax (Life Technologies, 0.3 μL/well). Twenty-four hourspost-transfection, cells were lysed and cDNA were generated (TaqManCells-to-CT Gene Expression kit, Life Technologies). AAT gene knockdownwas assessed by qRT-PCR with TaqMan chemistry-based assays (LifeTechnologies) for human AAT (Assay ID: Hs01097800_m1), normalized to theendogenous control, human cyclophilin A (PPIA, 4326316E). Of the 46siRNAs tested in vitro, five exhibited AAT knockdown of at least 80%.These were chosen for further analysis. Ten-point EC₅₀ curves weregenerated using the same cells and transfection conditions, with siRNAconcentrations ranging from 0.001-1 nM. Additionally, each of the fivemost efficacious canonical siRNAs was redesigned and synthesized as acorresponding mero RNAi trigger, UNA RNAi trigger and locked nucleicacid (LNA) RNAi trigger. The resultant RNAi triggers were again examinedby in vitro knockdown analysis, by both two-point concentration analysisat 1 nM and 0.1 nM and ten-point ED₅₀ determination. The mostefficacious of these were chosen for further in vivo studies. The mostpotent of these, SEQ ID 50/62 targeted position 1142-1160 in the AATmRNA and had an EC50 of 0.01 nM.

Serum Factor VII (F7) Activity Measurements.

Serum samples from animals were prepared by collecting blood intomicrocentrifuge tubes. F7 activity in plasma was measured with achromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason,Ohio) following manufacturer's recommendations. Absorbance ofcolorimetric development was measured using a Tecan Safire2 microplatereader at 405 nm.

Example 5. In Vivo Analysis of RNAi Trigger Efficacy in Mouse AATD Model

In order to evaluate the efficacy of candidate RNAi triggers in vivo,transgenic PiZ mouse model (PiZ mice) was used. PiZ mice harbor thehuman PiZ AAT mutant allele and model human AATD (Carlson et al. Journalof Clinical Investigation 1989). As noted above, AAT RNAi triggers wherechosen in silico for interaction with human and cynomolgus monkey AATbut not with rat or mouse AAT.

Cholesterol-targeted RNAi triggers were delivered to PiZ mice using MLPdelivery polymer. Each mouse received an intravenous (IV) injection intothe tail vein of 200-250 μL solution containing a dose of 8 or 2 mg/kgRNAi trigger+8 mg/kg MLP delivery polymer (1:1 or 0.25:1 w/w RNAitrigger:delivery polymer, respectively). Human AAT protein (hAAT) levelsin serum were monitored by assaying serum from the mice using an ELISAfor hAAT (Abcam) until hAAT expression levels returned to baseline. Fornormalization, AAT level for each animal at a time point was divided bythe pre-treatment level of expression in that animal (in this case atday 1) to determine the ratio of expression “normalized to day 1”.Expression at a specific time point was then normalized to the salinecontrol group by dividing the “normalized to day 1” ratio for anindividual animal by the mean “normalized to day 1” ratio of all mice inthe saline control group. This resulted in expression for each timepoint normalized to that in the control group. Experimental error isgiven as standard deviation.

mRNA quantitation. Isolation of RNA from PiZ mouse liver was performedas follows. At the time of euthanization, one to three sections of theliver were snap-frozen in 1.5 mL micro-centrifuge tubes using liquidnitrogen. One liver section from each mouse was transferred to 2 mL ofTRI Reagent RT (Molecular Research Center, Inc., Cincinnati, Ohio) in a15 mL conical tube. Total RNA was isolated following the manufacturer'srecommended protocol. Briefly, liver sections in TRI Reagent RT weretreated with a tissue homogenizer for approximately 30 sec. 1 mLhomogenate was added to 50 μL of 4-bromoanisole, mixed, and phases wereseparated by centrifugation. 0.25-0.5 mL of aqueous phase was removed,precipitated with isopropyl alcohol, and centrifuged. The resultantpellet was washed with 75% ethanol and suspended in 0.3-0.7 mLnuclease-free water.

Total RNA (˜500 ng) was reverse transcribed using the High Capacity cDNAReverse Transcription Kit (Life Technologies, Grand Island, N.Y.). ThecDNA was then diluted 1:5 and multiplex RT-qPCR was performed using 5′exonuclease chemistry with the commercially available FAM-labeled assayfor human alpha-1-antitrypsin (Assay ID Hs01097800_m1, LifeTechnologies), the VIC-labeled endogenous control assay for mousebeta-actin (Life Technologies) and VeriQuest Master Mix (Affymetrix,Santa Clara, Calif.). Gene expression data were analyzed using thecomparative C_(T) method of relative quantification (Livak andSchmittgen, 2001).

TABLE 7 PiZ mouse in vivo procedures. day procedure Day −7, −3, Bleedand serum isolation for hAAT ELISA −2 or −1 Day 1 a) Bleed and serumisolation for hAAT ELISA b) IV injection of samples Day 3 Bleed andserum isolation for hAAT ELISA Collect liver tissue (for RNA isolation)Day 8 Bleed and serum isolation for hAAT ELISA weekly Day 10 Bleed andserum isolation for hAAT ELISA; Collect liver tissue (for RNA isolation)Day 15, 22, 29, Weekly bleed and serum isolation for hAAT ELISA 36, 43

Example 6. Screening AAT siRNA RNAi Triggers and Time Course of AATKnockdown

Cholesterol-conjugated canonical siRNA RNAi triggers were administeredto PiZ mice as described above. Each mouse received a single intravenous(IV) dose of 2 mg/kg of RNAi trigger with 8 mg/kg of MLP deliverypolymer. Human AAT protein levels in serum were monitored for up to 29days. Knockdown levels and duration of response are shown in Table 8. Adecrease in hAAT serum protein level of greater than 95% was obtainedfollowing administration of SEQ ID 50/63 and SEQ ID 56/77.

TABLE 8 Serum hAAT protein levels in PiZ mice following administrationof 2 mg/kg siRNA with 8 mg/kg MLP delivery polymer. AAT levels werenormalized to day 1 and saline control. Serum hAAT normalized to controlgroup Treatment day −2 day 1 day 3 day 8 day 15 day 22 day 29 Saline1.00 ± 0.49 1.00 1.00 ± 0.35 1.00 ± 0.32 1.00 ± 0.17 1.00 ± 0.19 1.00 ±0.47 SEQ ID 50/63 1.03 ± 0.11 1.00 0.03 ± 0.01 0.02 ± 0.00 0.60 ± 0.421.30 ± 0.80 1.39 ± 0.38 SEQ ID 56/77 0.86 ± 0.23 1.00 0.22 ± 0.07 0.03 ±0.01 0.08 ± 0.01 0.58 ± 0.29 1.26 ± 0.37

Example 7. Screening AAT Mero RNAi Triggers and Time Course of AATKnockdown

Cholesterol-conjugated mero RNAi triggers were administered to PiZ miceas described above. Each mouse received a single intravenous (IV) doseof 8 or 2 mg/kg of RNAi trigger with 8 mg/kg of MLP delivery polymer.Human AAT protein levels in serum were monitored for up to 39 days.Knockdown levels and duration of response are shown in Table 10.

TABLE 9 Serum FVII levels in PiZ mice following administration of 8mg/kg mero RNAi triggers with 8 mg/kg MLP delivery polymer. FVII levelswere normalized to day 3 and saline control. Treatment FVII activitySaline 1.00 ± 0.06 SEQ ID 61/80 (FVII) 0.15 ± 0.20 SEQ ID 50/64/81 1.18± 0.17 SEQ ID 50/65/82 1.29 ± 0.26 SEQ ID 50/66/83 1.12 ± 0.47 SEQ ID56/76/89 1.24 ± 0.20 SEQ ID 55/72/86 1.33 ± 0.09

TABLE 10 Serum hAAT protein levels in PiZ mice following administrationof 8 mg/kg mero RNAi triggers with 8 mg/kg MLP delivery polymer. AATlevels were normalized to day 1 and saline control. Serum hAATnormalized to control group day −3, day −2 or Treatment day −1 day 1 day3 day 8 day 15 day 22 day 29 day 39 Saline 1.00 ± 0.10 1.00 1.00 ± 0.301.00 ± 0.08 1.00 1.00 1.00 SEQ ID 1.07 ± 0.08 1.00 0.74 ± 0.17 0.79 ±0.15 61/80 (FVII) 8 mg/kg SEQ ID 1.13 ± 0.03 1.00 0.11 ± 0.02 0.84 ±0.15 50/64/81 8 mg/kg SEQ ID 0.80 ± 0.14 1.00 0.07 ± 0.02 0.09 ± 0.071.34 ± 0.38 1.30 ± 0.17 1.11 ± 0.27 50/65/82 8 mg/kg SEQ ID 1.08 ± 0.161.00 0.10 ± 0.06 0.57 ± 0.21 50/66/83 8 mg/kg SEQ ID 1.10 ± 0.26 1.000.12 ± 0.04 0.07 ± 0.03 0.53 ± 0.01 1.15 ± 0.15 1.39 ± 0.35 56/76/89 8mg/kg SEQ ID 0.68 ± 0.16 1.00 0.22 ± 0.12 0.38 ± 0.10 0.45 ± 0.08 0.68 ±0.01 0.52 ± 0.07 0.65 ± 0.18 55/71/85 8 mg/kg SEQ ID 1.19 ± 0.02 1.000.37 ± 0.24 1.07 ± 0.04 55/72/86 8 mg/kg SEQ ID 0.69 ± 0.15 1.00 0.33 ±0.07 0.49 ± 0.07 0.60 ± 0.12 0.81 ± 0.16 0.73 ± 0.10 0.69 ± 0.1255/73/87 8 mg/kg SEQ ID 1.18 ± 0.23 1.00 0.36 ± 0.24 0.77 ± 0.28 1.71 ±0.20 1.65 ± 0.52 1.89 ± 0.61 53/68/84 2 mg/kg SEQ ID 0.77 ± 0.16 1.000.13 ± 0.04 0.79 ± 0.16 1.65 ± 0.14 1.39 ± 0.44 1.33 ± 0.46 55/74/88 2mg/kg

Example 8. In Vivo Screening AAT UNA RNAi Triggers and Time Course ofAAT Knockdown

Cholesterol-conjugated UNA RNAi triggers were administered to PiZ miceas described above. Each mouse received a single intravenous (IV) doseof 8 or 2 mg/kg of RNAi trigger with 8 mg/kg of MLP delivery polymer.Human AAT protein levels in serum were monitored for 40 days.

TABLE 11 Serum hAAT protein levels in PiZ mice following administrationof 8 or 2 mg/kg UNA RNAi triggers with 8 mg/kg MLP delivery polymer. AATlevels were normalized to day 1 and saline control. Serum hAATnormalized to control group day −3 or Treatment day −2 day 1 day 3 day 8day 15 day 22 day 29 day 39 Saline 1.00 ± 0.09 1.00 1.00 ± 0.11 1.00 ±0.09 1.00 ± 0.22 1.00 ± 0.10 1.00 ± 0.15 1.00 ± 0.16 SEQ ID 50/63 0.52 ±0.14 1.00 0.14 ± 0.03 0.03 ± 0.01 0.46 ± 0.13 0.74 ± 0.14 0.69 ± 0.160.77 ± 0.12 8 mg/kg^(a) SEQ ID 50/63 1.03 ± 0.11 1.00 0.03 ± 0.01 0.02 ±0.00 0.60 ± 0.42 1.29 ± 0.79 1.39 ± 0.38 2 mg/kg^(a) SEQ ID 51/63 0.88 ±0.04 1.00 0.15 ± 0.00 0.06 ± 0.00 0.41 ± 0.16 0.61 ± 0.08 0.65 ± 0.100.70 ± 0.06 8 mg/kg SEQ ID 52/63 0.81 ± 0.24 1.00 0.17 ± 0.03 0.03 ±0.01 0.07 ± 0.04 0.18 ± 0.07 0.31 ± 0.15 0.49 ± 0.19 8 mg/kg SEQ ID57/77 0.80 ± 0.10 1.00 0.19 ± 0.04 0.04 ± 0.02 0.47 ± 0.20 1.05 ± 0.281.72 ± 0.04 2 mg/kg SEQ ID 58/77 0.81 ± 0.19 1.00 0.09 ± 0.03 0.05 ±0.02 0.41 ± 0.24 0.59 ± 0.18 1.33 ± 0.43 2 mg/kg ^(a)canonical siRNAcontrol

A decrease in hAAT serum protein level of greater than 95% was obtainedfollowing administration of canonical siRNA SEQ ID 50/63 and UNA SEQ ID52/63. Maximum knockdown was observed 7 days after injection (day 8).Knockdown of greater than 80% reduction was sustained for more than 21days (day 22) with UNA SEQ ID 52/63. Knockdown persisted longer for theUNA RNAi triggers than for the canonical chol-siRNA of the same sequenceSEQ ID 50/63.

Example 9. Liver mRNA Analysis

AAT RNAi triggers were administered to PiZ mice as described above. Eachmouse received a single intravenous (IV) dose of 6 mg/kg of RNAi triggerwith 6 mg/kg of MLP delivery polymer. Liver hAAT mRNA production wasmeasured at days 3 and 10. Reduced mRNA levels correlated with decreasedserum hAAT protein levels, except that mRNA reduction preceded proteinreduction by a few days. The level of liver hAAT mRNA production wasmeasured at day 3 and day 10 following a single dose of SEQ ID 52/63with MLP delivery polymer in PiZ mice. A sustained decrease in liverhAAT mRNA levels was observed that correlated with the decrease observedin serum hAAT protein levels.

TABLE 12 Serum hAAT protein levels in PiZ mice following administrationof 6 mg/kg of SEQ ID 52/63 RNAi trigger or siLuc siRNA control with 6mg/kg of MLP delivery polymer. Serum hAAT levels were normalized to day1 and saline control. Serum hAAT normalized to day 1 Treatment day −2day 1 day 3 day 10 Saline 0.938 ± 0.168 1.00 1.077 ± 0.127 — siLuc 0.766± 0.219 1.00 1.110 ± 0.147 — SEQ ID 52/63 1.111 ± 0.605 1.00 0.326 ±0.021 — SEQ ID 52/63 0.483 ± 0.060 1.00 0.274 ± 0.072 0.105 ± 0.033

TABLE 13 Liver hAAT mRNA levels in PiZ mice following administration of6 mg/kg SEQ ID 52/63 RNAi trigger or siLuc siRNA control with 6 mg/kgMLP delivery polymer. AAT mRNA level is expressed relative to mouseβ-actin mRNA level. hAAT mRNA level Treatment day 3 day 10 Saline 1.00 ±0.15 — siLuc siRNA control 1.06 ± 0.20 — SEQ ID 52/63 0.025 ± 0.02 0.055 ± 0.02

Example 10. In Vivo Dose Response for SEQ ID 52/63 RNAi Trigger

Various amounts of UNA SEQ ID 52/63 were administered to PiZ mice asdescribed above. Each mouse received a single intravenous (IV) dose ofSEQ ID 52/63 with either 4 or 8 mg/kg of MLP delivery polymer. Human AATprotein levels in serum were monitored for 35 days. The level of hAATknockdown was largely dose dependent, in relation to both the dose ofSEQ ID 52/63 and dose of MLP delivery polymer (FIG. 8).

TABLE 14 Levels of serum hAAT in PiZ mice normalized to Day 1 and salinecontrol group mg/kg mg/kg MLP Serum hAAT normalized to Day 1 and controlSEQ ID delivery Day Day Day Day Day 52/63 polymer −3 Day 1 Day 3 Day 815 22 29 35 Saline control 1.00 ± 0.08 1.00 1.00 ± 0.02 1.00 ± 0.11 1.00± 0.04 1.00 ± 0.02 1.00 ± 0.06 1.00 ± 0.09 4 8 0.73 ± 0.13 1.00 0.20 ±0.04 0.05 ± 0.01 0.09 ± 0.01 0.28 ± 0.05 0.35 ± 0.05 0.81 ± 0.12 2 80.53 ± 0.08 1.00 0.17 ± 0.03 0.05 ± 0.00 0.10 ± 0.03 0.22 ± 0.03 0.40 ±0.06 0.63 ± 0.04 0.5 8 0.71 ± 0.03 1.00 0.20 ± 0.02 0.10 ± 0.03 0.19 ±0.05 0.55 ± 0.05 0.49 ± 0.09 0.71 ± 0.09 4 4 0.70 ± 0.17 1.00 0.27 ±0.03 0.13 ± 0.02 0.23 ± 0.07 0.58 ± 0.09 0.60 ± 0.08 0.92 ± 0.22 2 40.67 ± 0.02 1.00 0.25 ± 0.03 0.21 ± 0.07 0.32 ± 0.06 0.71 ± 0.14 0.66 ±0.06 0.70 ± 0.09 0.5 4 0.64 ± 0.10 1.00 0.27 ± 0.02 0.29 ± 0.09 0.43 ±0.01 0.73 ± 0.03 0.66 ± 0.05 0.97 ± 0.02

Example 11. In Vivo Dose Response for SEQ ID 52/63 RNAi Trigger

Various amounts of UNA SEQ ID 52/63 were administered to PiZ mice asdescribed above. Each mouse received a single intravenous (IV) dose ofSEQ ID 52/63 with either 2, 4 or 8 mg/kg of MLP delivery polymer. HumanAAT protein levels in serum were monitored for 36 days. Increasing doseof UNA generally led to increased level and duration of knockdown foreach level of MLP delivery polymer excipient used.

TABLE 15 Serum hAAT protein levels in PiZ mice following administrationof varying doses of SEQ ID 52/63 UNA RNAi triggers with varying doses ofMLP delivery polymer. AAT levels were normalized to day 1 and salinecontrol. mg/kg SEQ ID mg/kg Normalized serum hAAT levels 52/63 MLP day−7 day 1 day 8 day 15 day 20 day 29 day 36 Saline 1.00 ± 0.21 1.00 1.00± 0.14 1.00 ± 0.16 1.00 ± 0.12 1.00 ± 0.16 1.00 ± 0.13 2 2 0.91 ± 0.111.00 0.32 ± 0.24 0.88 ± 0.13 0.89 ± 0.18 1.01 ± 0.23 1.02 ± 0.15 4 21.27 ± 0.07 1.00 0.08 ± 0.03 0.68 ± 0.13 0.90 ± 0.15 1.07 ± 0.07 1.01 ±0.08 8 2 0.70 ± 0.15 1.00 0.09 ± 0.05 0.59 ± 0.16 0.74 ± 0.10 0.87 ±0.20 0.74 ± 0.08 2 4 0.90 ± 0.15 1.00 0.07 ± 0.04 0.50 ± 0.19 0.67 ±0.12 0.89 ± 0.06 0.94 ± 0.17 4 4 0.68 ± 0.07 1.00 0.03 ± 0.01 0.23 ±0.04 0.32 ± 0.05 0.66 ± 0.10 0.83 ± 0.06 8 4 0.70 ± 0.24 1.00 0.04 ±0.02 0.27 ± 0.05 0.35 ± 0.05 0.80 ± 0.20 1.00 ± 0.20 8 8 0.89 ± 0.541.00 0.02 ± 0.00 0.13 ± 0.06 0.16 ± 0.04 0.43 ± 0.08 0.78 ± 0.23

Example 12. Liver Histology in PiZ-Transgenic Mice Treated with SEQ ID52/63 RNAi Trigger

To further evaluate efficacy of hAAT knockdown in the liver,histological changes were assessed in liver samples from male PiZ micefollowing administration of SEQ ID 52/63 RNAi trigger with MLP deliverypolymer. UNA SEQ ID 52/63 was administered to PiZ mice as describedabove. Each mouse received a biweekly administration of an intravenous(IV) dose of 8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLP delivery polymerfor 8 weeks. Mice were bled weekly to monitor hAAT levels in serum andwere sacrificed on day 57 after administration of SEQ ID 52/63 with MLPdelivery polymer. Liver samples were harvested and fixed in 10%neutral-buffered formalin and embedded in paraffin. Inflammatoryinfiltration was assessed by H&E staining. The PiZ mice injectedbiweekly with 8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLP delivery polymerfor 8 weeks had normal morphology, no detectably inflammatory infiltrateand very rare, small Z-hAAT globules. PiZ mice injected biweekly withsaline had significant globule accumulation as well as inflammatoryinfiltration around damaged or dead hepatocytes. Aggregation of Z-hAATwas visualized by performing diastase-resistant periodic acid Schiff(PAS-D) staining on liver sections. Diastase digestion of glycogen priorto performing a PAS stain allows positive staining of Z-AAT proteinaccumulation, or “globules”. PiZ mice that received four biweeklyintravenous (IV) doses of 8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLPdelivery polymer over the course of 8 weeks showed a decrease inintracellular AAT globules compared to PiZ mice receiving saline or LucUNA RNAi trigger 59/78 control injections (luciferase RNAi trigger:dTCfgAfaGfUUNAAfcUfcAfgCfgUfaAfgdTsdT, SEQ ID 59;(Chol-TEG)uAuCfuUfaCfgCfuGfaGfuAfcUfuCfgAf(invdT), SEQ ID 78). Thenumber of globules, the size of the globules and the area of the livercovered by globules was digitally quantitated from liver specimensstained with PAS-D. AAT-UNA treated mice had 85% fewer globules, 85%smaller globules, and 96% less area of the liver covered with globulesthan saline-injected controls (FIG. 10).

Example 13. Analysis of Soluble and Insoluble Z-hAAT Protein in PiZMouse Liver Tissue

Homogenized liver tissue from PiZ mice treated with SEQ ID 52/63 RNAieffector was further analyzed to determine if both soluble Z-hAAT,expected to be mostly monomeric protein, and insoluble polymers ofZ-hAAT were effectively reduced. A modified western blot protocol wasused to separate the soluble and insoluble Z-hAAT fractions undernon-denaturing conditions as previously described (Mueller et al.Molecular Therapy 2012). PiZ mice given four biweekly intravenous (IV)doses of 8 mg/kg SEQ ID 52/63 with 8 mg/kg of MLP delivery polymer for 8weeks showed a 99% reduction in soluble and 79% reduction in insolubleZ-hAAT, compared to PiZ mice given four biweekly intravenous (IV) dosesof saline (FIG. 11).

TABLE 16 Average levels of soluble and insoluble Z-hAAT protein in liverlysate of PiZ mice normalized to saline-injected mice Soluble Insolublepolymer Number (normalized to (normalized to Treatment animals salinecontrol) saline control) Baseline (5 weeks old) 3 0.866 ± 0.105 0.478 ±0.083 Saline (13 weeks old) 7 0.992 ± 0.138 1.010 ± 0.309 Luc-UNA (13weeks old) 3 1.630 ± 0.162 1.192 ± 0.152 SEQ ID 53/63 (13 weeks 10 0.004± 0.013 0.209 ± 0.103 old)

Example 14. In Vivo Duration of Response from Single Injection of SEQ ID52/63 RNAi Trigger in PiZ Mice

A single IV dose of saline, 8 mg/kg Luc-UNA+8 mg/kg MLP deliverypolymer, or 8 mg/kg AAT RNAi trigger SEQ ID 52/63+8 mg/kg MLP deliverypolymer was administered to 6 month old female PiZ mice as describedabove. Human AAT protein levels in serum were monitored for 29 days. Atthe indicated times, blood samples were collected and assayed for hAATby ELISA. Day 1 samples were collected prior to trigger administration.For mRNA analysis, 3-4 mice were euthanized at each of days 3, 8, 15,22, and 29. For euthanized mice, cardic stick were performed for serumisolation for AAT ELISA (200 μl serum). Half of the left lateral liverlobe was collected and snap-freeze in liquid nitrogen for RNA isolation.The remainder of the left lobes were embedded into paraffin blocks forPAS-D staining with hematoxylin as counter-stain. Serum hAAT levels inmice given AAT RNAi trigger SEQ ID 52/63 were 95% reduced on day 8 andremained reduced to day 29, at which time they were 79% reduced. Micegiven AAT RNAi trigger SEQ ID 52/63 were euthanized at either day 3, 8,15, 22 or 29. Mice given saline or Luc-RNAi trigger (SEQ ID 59/78) wereeuthanized on day 29. Levels of hAAT mRNA in the livers were measured byRT-qPCR. The hAAT mRNA in mice given UNA SEQ ID 52/63 was reduced by 97%on day 3 and remained reduced on day 29, at which time levels were 56%reduced. The size of the globules and the area of the liver covered byglobules was digitally quantitated from liver specimens stained withPAS-D. AAT-UNA treated mice had 70% smaller globules at day 15 and 62%smaller globules at day 29. The area of the liver covered with globuleswas 83% reduced on day 15 and 72% reduced on day 29 (FIG. 12).

TABLE 17 Serum hAAT levels in PiZ mice following administration of oneinjection of saline, Luc-RNAi trigger SEQ ID 59/78, or AAT RNAi triggerSEQ ID 52/63. Serum hAAT normalized to day 1 and controls Day Day DayDay Day euthanized Treatment −2 Day 1 Day 3 Day 8 15 22 29 Day 29 Saline1.000 ± 0.239 1.000 1.000 ± 0.368 1.000 ± 0.235 1.000 ± 0.097 1.000 ±0.177 1.000 ± 0.272 Day 29 8 mg/kg MLP 1.268 ± 0.143 1.000 0.994 ± 0.1531.090 ± 0.092 0.890 ± 0.080 1.171 ± 0.095 0.797 ± 0.074 deliverypolymer + 8 mg/kg Luc-UNA Day 3 8 mg/kg MLP 1.068 ± 0.070 1.000 0.255 ±0.040 — — — — Day 8 delivery 0.895 ± 0.129 1.000 0.184 ± 0.025 0.054 ±0.004 — — — Day 15 polymer +  0.66 ± 0.121 1.000 0.243 ± 0.060 0.066 ±0.023 0.095 ± 0.063 — — Day 22 8 mg/kg 0.779 ± 0.280 1.000 0.202 ± 0.0540.044 ± 0.010 0.056 ± 0.011 0.100 ± 0.039 — Day 29 AAT-UNA 0.653 ± 0.1021.000 0.238 ± 0.062 0.052 ± 0.015 0.057 ± 0.016 0.103 ± 0.032 0.209 ±0.071

TABLE 18 Relative hAAT mRNA levels in PiZ mice following administrationof one injection of saline, Luc-RNAi trigger SEQ ID 59/78, or AAT RNAitrigger SEQ ID 52/63. Average relative Low High Treatment day mRNA levelvariance variance Saline 29 1.000 0.072 0.078 8 mg/kg Luc-RNAi trigger +29 1.031 0.090 0.098 8 mg/kg MLP delivery polymer 8 mg/kg mg/kg SEQ ID52/63 + 3 0.030 0.007 0.009 8 mg/kg MLP delivery polymer 8 0.032 0.0140.024 15 0.158 0.060 0.096 22 0.221 0.033 0.038 29 0.439 0.057 0.066

Example 15. Alpha-1 Antitrypsin (AAT) Knockdown in Primate Following AATRNA Trigger Molecule Delivery by MLP Delivery Polymer

MLP delivery polymer and RNAi trigger were made and combined in apharmaceutically acceptable buffer as described above. On day 1,cynomolgus macaque (Macaca fascicularis) primates (male and female, 3 to9 kg) were co-injected with MLP delivery polymer and AAT UNA RNAitrigger SEQ ID 52/63 at different dose combinations. The dosecombinations injected were: 2.0 mg/kg MLP delivery polymer+4.0 RNAitrigger (n=3), 3 mg/kg MLP delivery polymer+1.5 mg/kg RNAi trigger(n=2), 3.0 mg/kg MLP delivery polymer+3.0 mg/kg RNAi trigger (n=3), 3.0mg/kg MLP delivery polymer+6.0 mg/kg RNAi trigger (n=2), 6.0 mg/kg MLPdelivery polymer+12 mg/kgRNAi trigger (n=3) (0.050 s conversion factorused to determine RNAi trigger concentration) and 12 mg/kg MLP deliverypolymer+6.0 mg/kg RNAi trigger (n=12). For each injection the MLPdelivery polymer+RNAi trigger (2 ml/kg) was injected into the saphenousvein using a 22 to 25 gauge intravenous catheter. At the indicated timepoints, blood samples were drawn and analyzed for AAT and toxicitymarkers. Blood was collected from the femoral vein and primates werefasted overnight before all blood collections. Blood tests for bloodurea nitrogen (BUN), alanine transaminase (ALT), aspartateaminotransferase (AST), and creatinine were performed on an automatedchemistry analyzer at Meriter laboratories or BASi. AAT levels weredetermined on a Cobas Integra 400 (Roche Diagnostics) according to themanufacturer's recommendations. Significant knockdown of AAT wasobserved at all dose combinations. No toxicity was observed at doseswith 2 mg/kg, 3 mg/kg or 6 mg/kg of MLP but at 12 mg/kg MLP there wereelevations in liver enzymes (ALT and AST) as well as BUN and creatinineafter injection.

TABLE 19 Percent AAT Knockdown in NHPs SEQ ID MLP 52/63 Day (mg/kg)(mg/kg) Pretest 2 3 8 11 15 22 26 29 33 36 43 47 50 2.0 4.0 0 11 27 6373 81 85 — 80 — 3.0 1.5 0 16 30 59 70 74 76 — 74 — 70 63 — 51 3.0 3.0 015 29 63 74 82 88 — 85 — 3.0 6.0 0 10 25 63 74 82 6.0 12.0 0 18 30 6112.0 6.0 0 3 19 60 84 88 — 91 — 86 — — 76

TABLE 20 Urea nitrogen (mg/dL) SEQ ID 52/63 MLP (mg/kg) (mg/kg) PretestDay 2 Day 3 Day 8 2.0 4.0 20 21 20 20 3.0 1.5 18 21 18 20 3.0 3.0 18 1818 16 3.0 6.0 22 22 23 22 6.0 12.0 16 18 15 12.0 6.0 17 39 42 20

TABLE 21 Creatinine (mg/dL) SEQ ID 52/63 MLP (mg/kg) (mg/kg) Pretest Day2 Day 3 Day 8 2.0 4.0 0.88 0.9 0.94 0.89 3.0 1.5 0.81 0.8 0.85 0.81 3.03.0 0.7 0.77 0.83 0.72 3.0 6.0 0.82 0.92 0.99 0.84 6.0 12.0 0.6 0.660.67 12.0 6.0 0.58 1.88 1.32 0.7

TABLE 22 Alanine transaminase (U/L) SEQ ID 52/63 MLP (mg/kg) (mg/kg)Pretest Day 2 Day 3 Day 8 2.0 4.0 44 52 58 47 3.0 1.5 56 62 65 56 3.03.0 34 54 54 35 3.0 6.0 43 54 53 39 6.0 12.0 41 52 47 12.0 6.0 48 81 6033

TABLE 23 Aspartate aminotransferase (U/L) MLP delivery SEQ ID 52/63polymer (mg/kg) (mg/kg) Pretest Day 2 Day 3 Day 8 2.0 4.0 28 48 54 303.0 1.5 49 58 54 35 3.0 3.0 27 67 57 28 3.0 6.0 35 58 51 29 6.0 12.0 3446 33 12.0 6.0 29 249 89 55

Example 16. Repeat Administration

Cynomolgus macaque primates were given five doses of RNAi trigger+MLPdelivery polymer at six week intervals. Each dose contained MLP deliverypolymer and AAT-RNAi trigger SEQ ID 52/63 at a 1:2 weight to weightratio of the MLP to RNAi trigger. The first injection was on day 1. Thedose combinations injected were: 2.0 mg/kg MLP delivery polymer+4.0 RNAitrigger (n=2) and 3 mg/kg MLP delivery polymer+6 mg/kg RNAi trigger(n=2). Blood was collected at intervals throughout the study and AATlevels were measured from the serum as described. Repeat dosing at sixweek intervals reduced serum AAT levels by approximately 80-90% from twoto thirty weeks after the first treatment of 3 mg/kg MLP deliverypolymer+6 mg/kg RNAi trigger in the primates. Serum AAT was reduced by80% following the first treatment of primates with 2.0 mg/kg MLPdelivery polymer+4.0 RNAi trigger and by 85% following the fourthtreatment. Serum AAT levels measured six weeks after each treatment with2.0 mg/kg MLP delivery polymer+4.0 RNAi trigger rebounded less with eachadditional treatment (FIG. 13).

The invention claimed is:
 1. An RNA interference (RNAi) trigger moleculecapable of inhibiting the expression of an alpha-1 antitrypsin genewherein: (i) the RNAi trigger molecule comprises a sense sequence and anantisense sequence, wherein said antisense sequence comprises in ordernucleotides 1-18 of SEQ ID NO: 5, (ii) the sense sequence and/or theantisense sequence further comprise a 3′ and/or 5′ extension of 1-5nucleotides in length, (iii) the 3′ end nucleotide of the sense sequenceis an inverted (3′ to 3′ linked) deoxynucleotide, and, (iii) theextended antisense sequence is capable of inhibiting the expression ofan alpha-1 antitrypsin gene.
 2. The RNAi trigger molecule of claim 1,wherein the sense strand or the antisense strand further comprises a 3′extension of 1-5 nucleotides in length.
 3. The RNAi trigger molecule ofclaim 2, wherein the 3′ extension of the antisense strand comprises dTdTor dTsdT.
 4. The RNAi trigger molecule of claim 2, wherein the 3′extension of the sense strand comprises At(invdT).
 5. The RNAi triggermolecule of claim 1, wherein the sense strand or the antisense strandfurther comprises a 5′ extension of 1-5 nucleotides in length.
 6. TheRNAi trigger molecule of claim 5, wherein the 5′ extension of theantisense strand comprises dT.
 7. The RNAi trigger molecule of claim 5,wherein the 5′ extension of the sense strand comprises UAU or uAu. 8.The RNAi trigger molecule of claim 1, wherein a targeting moiety isconjugated to the 5′ end of the sense strand.
 9. The RNAi triggermolecule of claim 8 wherein the targeting moiety comprises a cholesterylgroup.
 10. The RNAi trigger molecule of claim 9 wherein the targetingmoiety comprises a cholesterol-triethylene glycol group.
 11. The RNAitrigger molecule of claim 1, wherein the sense sequence and an antisensesequence form a sequence pair of SEQ ID NOs: 5/12.
 12. The RNAi triggermolecule of claim 2, wherein the sense sequence and an antisensesequence form sequence pairs or meroduplexes selected from the groupconsisting of SEQ ID NOs: 16/36, 19/37/49, and 19/38.
 13. The RNAitrigger molecule of claim 1 wherein the sense strand or antisense strandcontains one or more modified nucleotide or nucleotide mimics.
 14. TheRNAi trigger molecule of claim 13, wherein the sense sequence and anantisense sequence form sequence pairs selected from the groupconsisting of SEQ ID NOs: 56/75, and 56/77.
 15. The RNAi triggermolecule of claim 13, wherein the sense sequence and an antisensesequence form a sequence meroduplex of SEQ ID NOs: 56/76/89.
 16. TheRNAi trigger molecule of claim 13 wherein the antisense sequencecontains at least one 2′,3′-seco RNA nucleotide mimic.
 17. The RNAitrigger molecule of claim 16, wherein the sense sequence and anantisense sequence form sequence pairs selected from the groupconsisting of SEQ ID NOs: 57/77, and 58/77.
 18. The RNAi triggermolecule of claim 13, wherein modified nucleotide is selected from thegroup consisting of: 2′-O-methyl modified nucleotide, nucleotidecomprising a 5′-phosphorothioate group, 2′-deoxy-2′-fluoro modifiednucleotide, 2′-deoxy-modified nucleotide, locked nucleotide, abasicnucleotide, deoxythymidine, inverted deoxythymidine, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide,phosphorothioate linked nucleotide, and non-natural base comprisingnucleotide.
 19. A pharmaceutical composition comprising the RNAi triggeracid molecule as defined in claim 1 and a MLP delivery polymer.
 20. Thepharmaceutical composition of claim 19 further comprising apharmaceutically acceptable carrier, stabilizer and/or diluent.
 21. Amethod for inhibiting the expression of an AAT gene in a cell, a tissue,or an organism comprising introducing into a cell, tissue, or organismthe RNAi trigger molecule as defined in claim
 1. 22. The method of claim21 wherein inhibiting expression of AAT gene in an organism treats,prevents, or manages a pathological condition or disease caused byalpha-1 antitrypsin deficiency.
 23. The method claim 22 wherein thepathological condition and disease caused by alpha-1 antitrypsindeficiency is selected from the group consisting of: chronic hepatitis,cirrhosis, hepatocellular carcinoma, and fulminant hepatic failure.